Methods, compositions and kits for treating a subject using a recombinant neutralizing binding protein

ABSTRACT

Methods, compositions and kits are provided for treating a subject exposed to or at risk for exposure to a disease agent using a pharmaceutical composition including at least one recombinant binding protein or a source of expression of the binding protein, wherein the binding protein neutralizes a disease agent that is a toxin, for example a  Clostridium difficile  toxin, a Shiga toxin, a ricin toxin, or an anthrax toxin.

RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.15/191,739, filed Jun. 24, 2016, which is a continuation ofInternational PCT Application No. PCT/US14/72340, filed Dec. 24, 2014,which claims priority to U.S. provisional application 61/920,825, filedDec. 26, 2013, the contents of each of which are incorporated byreference herein in their entireties. International PCT Application No.PCT/US14/72340 is related to prior U.S. utility application U.S. Ser.No. 13/566,524, filed Aug. 3, 2012, now U.S. Pat. No. 9,023,352, issuedMay 5, 2015, and to U.S. provisional application 61/809,685, filed Apr.8, 2013, the entire contents of both of which are incorporated byreference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant AI057159awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates to, in part, compositions, methods, andkits using a recombinant neutralizing binding protein for treating asubject at risk for exposure or exposed to a disease agent.

BACKGROUND

Clostridium difficile infections cause serious disease with increasingincidence worldwide. The infections occur primarily in hospitals andlongterm care facilities in patients receiving prolonged antibiotictreatments. The disease symptoms may result from production and releaseby C. difficile organisms of two toxins, TcdA and TcdB in the colon.

A need exists for generating high affinity binding agents that treatboth routine incidents of disease and pandemics, and efforts to discoverand produce these agents are underway. The production of antibodies andtheir storage is a costly and lengthy process. In fact, development of asingle antibody therapeutic agent often requires years of clinicalstudy. Yet multiple, different therapeutic antibodies are necessary forthe effective treatment of patients exposed to a disease agent, aninfection outbreak or a bio-terrorist assault. Developing and producingmultiple antibodies that can bind to different targets (e.g. microbialpathogens, viral pathogens, toxins, and cancer cells) is often adifficult task because it involves separately producing, storing andtransporting multiple antibodies for each pathogen or toxin. Productionand stockpiling a sufficient amount of antibodies to protect largepopulations is a challenge and currently has not been achieved. Theshelf life of antibodies is often relatively short (e.g., weeks ormonths), and accordingly freshly prepared batches of antibodies have tobe produced to replace the expiring antibodies.

Accordingly, there is a need for a cost effective and efficient way toprovide alternatives to current therapeutic agents. Further a needexists for alternative therapeutics that are easier to develop andproduce, have a longer shelf life, and bind as a single agent tomultiple targets on the same disease agent, as well as to differentdisease agents.

SUMMARY

An aspect of the invention provides a pharmaceutical composition fortreating a subject exposed to at least one disease agent, such that thepharmaceutical composition includes at least one recombinant bindingprotein that neutralizes the one or more disease agents and treats thesubject for exposure to the disease agent, such that the binding proteinincludes at least one disease agent binding domain amino acid sequence,for example, selected from the group of:

(SEQ ID NO: 174) QVQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQ; (SEQ ID NO: 164)QLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQ; (SEQ ID NO: 165)QVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ; and (SEQ ID NO: 166)QVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQ.

In certain embodiments of the pharmaceutical composition comprises SEQID NO:167

(SEQ ID NO: 167) METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLSGAPVPYPDPLEPRAAAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSAAAQLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSALVGGGGSGGGGSGGGGSLQAMAAAQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQAIAGGGGSGGGGSGGGGSLQGQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQGAPVPYPDPLEPRGGGSDICLPRWGCL WED,or variants thereof.

In some embodiments, the pharmaceutical composition comprises a dimer ofone or more of a C. difficile A toxin-binding protein and a C. difficileB toxin-binding protein. In certain embodiments, the pharmaceuticalcomposition comprises a dimer of AH3 and AA6 or 5D and E3. In certainembodiments, the pharmaceutical composition comprises the amino acidssequences of SQ ID NOs: 171 and/or 172. In some embodiments, thepharmaceutical composition comprises a tetramer of one or more of a C.difficile A toxin-binding protein and a C. difficile B toxin-bindingprotein. In certain embodiments, the pharmaceutical compositioncomprises a tetramer of AH3, AA6, 5D, and E3. In certain embodiments,the pharmaceutical composition comprises the amino acids sequence of SEQID NO: 173. In various embodiments, the dimers or tetramers furthercomprise a linker of SEQ ID NO: 55.

In some embodiments, the present compositions, methods, and kits includethe following sequence:

(SEQ ID NO: 171, dimer of AH3-AA6)QGVQSQLQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKP Q.

In some embodiments, the present compositions, methods, and kits includethe following sequence:

(SEQ ID NO: 172, dimer of 5D-E3)QGVQSQLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ.

In some embodiments, the present compositions, methods, and kits includethe following sequence:

(SEQ ID NO: 173, tetramer of AH3, 5D, E3, AA6)QGVQSQLQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQGGGGSGGGGSGGGGSQGVQSQLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSE PKTPKPQ.

In certain embodiments of the pharmaceutical composition, the bindingprotein includes at least one Vh domain corresponding to heavychain-only camelid antibodies. For example, the recombinant bindingprotein includes at least three Vh disease agent-binding domains. Incertain embodiments, the three disease agent binding domains include atleast two of the amino acid sequences selected from the group of: SEQ IDNO: 174, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167,SEQ ID NO: 171, SEQ ID NO: 172, and, SEQ ID NO: 173 or variants thereof.In certain embodiments, each of the disease agent binding domains isseparated from another disease agent binding site by flexible spaceramino acid sequences. For example, the flexible spacer amino acidsequence includes amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 55),or a variant thereof. In certain embodiments, the recombinant bindingprotein includes a plurality of disease agent binding domains that bindand neutralize toxin A of Clostridium difficile (TcdA). In certainembodiments, the recombinant binding protein includes a plurality ofdisease agent binding domains that bind and neutralize toxin B ofClostridium difficile (TcdB).

In some embodiments, the pharmaceutical composition comprises a dimer ofone or more of a C. difficile A toxin-binding protein and a C. difficileB toxin-binding protein. In certain embodiments, the pharmaceuticalcomposition comprises a dimer of AH3 and AA6 or 5D and E3. In certainembodiments, the pharmaceutical composition comprises the amino acidssequences of SEQ ID NOs: 171 and/or 172. In some embodiments, thepharmaceutical composition comprises a tetramer of one or more of a C.difficile A toxin-binding protein and a C. difficile B toxin-bindingprotein. In certain embodiments, the pharmaceutical compositioncomprises a tetramer of AH3, AA6, 5D, and E3. In certain embodiments,the pharmaceutical composition comprises the amino acids sequence of SEQID NO: 173. In certain embodiments, the pharmaceutical compositioncomprises the amino acids sequence of SEQ ID NO: 167. In variousembodiments, the dimers or tetramers further comprise a linker of SEQ IDNO: 55.

In an embodiment of the pharmaceutical composition, the recombinantbinding protein includes an epitope tag amino acid sequence. Forexample, the recombinant binding protein includes a plurality of copiesof epitope tag SEQ 11) NO: 15, or a variant thereof. In an embodiment ofthe pharmaceutical composition, the recombinant binding protein carboxylterminus includes an albumin binding domain amino acid sequence. Forexample, the albumin binding domain comprises amino acid sequenceDICLPRWGCLWED (SEQ ID NO: 168), or a variant thereof. In an embodimentof the pharmaceutical composition, the recombinant binding protein aminoterminus includes an E. coli thioredoxin. In certain embodiments, therecombinant binding protein has a cleavage site between the E. colithioredoxin protein amino acid sequence and an adjacent disease agentbinding domain.

In certain embodiments of the pharmaceutical composition, the diseaseagent includes a first and second non-identical disease agent, and thebinding protein binds and neutralizes the first and the second ofdisease agents. In certain embodiments of the pharmaceuticalcomposition, the disease agent includes a Clostridium difficile toxin.

An aspect of the invention provides a pharmaceutical composition fortreating a subject at risk for exposure to at least one disease agent,the pharmaceutical composition including: a source of expression of arecombinant disease agent binding protein for neutralizing the diseaseagent and treating the subject for exposure, wherein the source ofexpression comprises nucleotide sequence:

(SEQ ID NO: 169) ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCA, or a variant thereof.

An aspect of the invention provides a kit for treating a subject exposedto or at risk for exposure to a disease agent including: a unit dosageof a pharmaceutical composition for treating a subject at risk forexposure to at least one disease agent such that, the pharmaceuticalcomposition includes: at least one recombinant binding protein thatneutralizes the disease agent thereby treating the subject for exposureto the disease agent, wherein the binding protein comprises at least oneamino acid sequence selected from the group of: SEQ ID NO: 174, SEQ IDNO: 164, SEQ ID NO: 165, SEQ ID NO: 166, and SEQ ID NO: 167, or variantsthereof; a container; and instructions for use. In certain embodiments,the pharmaceutical composition includes a pharmaceutically acceptableexcipient. In certain embodiments of the kit, the disease agent includesa Clostridium difficile toxin. In certain embodiments of the kit, thedisease agent comprises a first and a second non-identical diseaseagent, and the binding protein binds and neutralizes the first and thesecond disease agent. In certain embodiments of the kit, thepharmaceutical composition includes a plurality of the disease agentbinding domains that bind and neutralize toxin A of Clostridiumdifficile (TcdA). In certain embodiments of the kit, the pharmaceuticalcomposition includes a plurality of the disease agent binding domainsthat bind and neutralize toxin B of Clostridium difficile (TcdB). Insome embodiments, the kit comprises a dimer of one or more of a C.difficile A toxin-binding protein and a C. difficile B toxin-bindingprotein. In certain embodiments, the kit comprises a dimer of AH3 andAA6 or 5D and E3. In certain embodiments, the kit comprises the aminoacids sequences of SQ ID NOs: 171 and/or 172. In some embodiments, thekit comprises a tetramer of one or more of a C. difficile Atoxin-binding protein and a C. difficile B toxin-binding protein. Incertain embodiments, the kit comprises a tetramer of AH3, AA6, 5D, andE3. In certain embodiments, the kit comprises the amino acids sequenceof SEQ ID NO: 173. In various embodiments, the dimers or tetramersfurther comprise a linker of SEQ ID NO: 55.

An aspect of this invention provides a method for treating a subject atrisk for exposure to at least one disease agent, such that the methodincludes administering to the subject a source of expression of arecombinant disease agent binding protein, wherein the source ofexpression of the binding protein is a nucleotide sequence encoding thebinding protein, the nucleotide sequence comprising SEQ ID NO: 169, or avariant thereof; and measuring neutralization of the disease agent orplurality of disease agents by the binding protein.

An aspect of the invention provides a pharmaceutical composition fortreating a subject exposed to at least one disease agent, thepharmaceutical composition including: at least one recombinant bindingprotein that neutralizes the disease agent and treats the subject forexposure to the disease agent, such that the binding protein includes atleast one disease agent binding domain amino acid sequence selected fromthe group of: SEQ ID NO: 174, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:166, and SEQ ID NO:167; and further includes amino acid sequence SEQ IDNO: 168, or variants thereof.

An aspect of the invention provides a pharmaceutical composition fortreating a subject exposed to at least one disease agent, thepharmaceutical composition including at least one recombinant bindingprotein that neutralizes the disease agent and treats the subject forexposure to the disease agent, such that the binding protein includes atleast one disease agent binding domain amino acid sequence selected fromthe group of: SEQ ID NO: 174, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:166, and SEQ ID NO:167; and further including amino acid sequence SEQ IDNO: 53, or variants thereof.

In some embodiments, there is provided a method of treating orpreventing a C. difficile infection (CDI) or associated disease asdescribed herein comprising administering to a patient in need thereofan effective amount of a pharmaceutical composition comprising at leastone recombinant binding protein comprising at least one disease agentbinding domain amino acid sequence selected from SEQ ID NO: 174, SEQ IDNO: 164, SEQ ID NO: 165, SEQ ID NO: 166 SEQ ID NO: 167, SEQ ID NO: 171,SEQ ID NO: 172, and SEQ ID NO: 173 or variants thereof. In someembodiments, the recombinant binding protein comprising at least onedisease agent binding domain comprises a recombinant camelidheavy-chain-only antibody (VHH). In some embodiments, the patient inneed of such treatment is receiving or will receive treatment with oneor more antibiotics.

The sequence listing material in computer readable form ASCII text file(220 kilobytes) created 05/26/2016 entitled “34724171_SeqListing_ST25”,containing sequence listings numbers 1-174, has been electronicallyfiled herewith and is incorporated by reference herein in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A-FIG. 1 E are nucleotide sequences of scFv#2 (SEQ ID NO: 1),scFv#3 (SEQ ID NO: 3), scFv#7 (SEQ ID NO: 5), scFv#8 (SEQ ID NO: 7),scFv#21 (SEQ ID NO: 9), scFv#E (SEQ ID NO: 11), and amino acid sequencesof scFv#2 (SEQ ID NO: 2), scFv#3 (SEQ ID NO: 4), scFv#7 (SEQ ID NO: 6),scFv#8 (SEQ ID NO: 8), scFv#21 (SEQ ID NO: 10), scFv#E (SEQ ID NO: 12).

FIG. 2 is the nucleotide sequence of scFv#7-2E (SEQ ID NO: 13) and theamino acid sequence of scFv#7-2E (SEQ ID NO: 14).

FIG. 3 A-FIG. 3 C are the nucleotide sequences of BoNT/A holotoxinbinding VHHs including JDA-D12 (SEQ ID NO: 19), JDQ-A5 (SEQ ID NO: 21),JDQ-B5 (SEQ ID NO: 23), JDQ-C2 (SEQ ID NO: 25), JDQ-F12 (SEQ ID NO: 27),JDQ-G5 (SEQ ID NO: 29), JDQ-H7 (SEQ ID NO: 31), and BoNT/B holotoxinbinding VHHs including JEQ-A5 (SEQ ID NO: 33), JEQ-H11 (SEQ ID NO: 35).The figures also show the corresponding amino acid sequences of BoNT/Aholotoxin binding VHHs including JDA-D12 (SEQ ID NO: 20), JDQ-A5 (SEQ IDNO: 22), JDQ-B5 (SEQ ID NO: 24), JDQ-C2 (SEQ ID NO: 26), JDQ-F12 (SEQ IDNO: 28), JDQ-G5 (SEQ ID NO: 30), JDQ-H7 (SEQ ID NO: 32), and BoNT/Bholotoxin binding VHHs including JEQ-A5 (SEQ ID NO: 34), JEQ-H11 (SEQ IDNO: 36).

FIG. 4 A is a set of three nucleotide sequences of VHHs identified asBoNT/A binders that were experimentally shown to bind to the sameepitope, and the set of three corresponding VHH amino acid sequences.The VHH sequences are DQ-B5 (SEQ ID NO: 23), JDO-E9 (SEQ ID NO: 37), andJDQ-B2 (SEQ ID NO: 39), and the corresponding VHH amino acid sequencesare JDQ-B5 (SEQ ID NO: 24), JDO-E9 (SEQ ID NO: 38), and JDQ-B2 (SEQ IDNO: 40).

FIG. 4 B is a set of two nucleotide sequences of VHHs identified asBoNT/A binders that were experimentally shown to bind to the sameepitope, and the set of two corresponding VHH amino acid sequences. TheVHH sequences are JDQ-05 (SEQ ID NO: 41), and JDQ-F9 (SEQ ID NO: 43),and, the corresponding VHH amino acid sequences are JDQ-05 (SEQ ID NO:42), and JDQ-F9 (SEQ ID NO: 44).

FIG. 5 is a schematic drawing of a phylogenetic tree comparing thehomology between BoNT/A binding VHHs within the JDQ-B5 competition group(which compete for binding, thus bind the same epitope) in comparison tocontrol alpaca VHHs.

FIG. 6 is a schematic drawing of binding agent VHHs that are produced indifferent formats including formats in which the binding agents arefused to one or more E-tags or as fusion proteins.

FIG. 7 is a drawing of a single-tagged heterodimeric binding protein(exemplary VHHs) binding to the disease agent, a toxin, and leading todecoration of the toxin with two anti-tag monoclonal antibodies (mAbs).

FIG. 8 is a drawing of a double-tagged binding protein (here shown areVHHs) a heterodimeric binding to the disease agent, toxin, and leadingto decoration of the toxin with four anti-tag mAbs.

FIG. 9 A-FIG. 9 B are a set of Meyer-Kaplan survival plots thatdouble-tagged heterodimer E/H7/B5/E and the anti-tag mAb completelyprotected subjects from 1,000-fold and 1,000-fold the median lethal doseof a Botulinum neurotoxin serotype A toxin.

FIG. 9 A is a Meyer-Kaplan survival plot showing percent (%) of micesurviving over a period of time (days) after receiving 1,000-fold themedian lethal dose (LD₅₀) of a Botulinum neurotoxin serotype A (BoNT/A)and each of combinations of the following binding agents: H7 and B5 VHHheterodimer with a single epitopic tag (tag or E-tag) and an anti-E-tagmAb (H7/B5/E+anti-E mAb); H7 and B5 VHH monomers each with an E-tag andan anti-E-tag mAb (H7/E+B5/E+anti-E mAb); H7 and B5 VHH heterodimer withtwo E-tags and an anti-E-tag mAb (E/H7/B5/E+anti-E mAb) and a control(the toxin alone). The data show that administration of heterodimerE/H7/B5/E and anti-E mAb resulted in survival of subjects for sevendays.

FIG. 9 B is a Meyer-Kaplan survival plot showing percent (%) of subjectssurviving over a period of time (days) after receiving 10,000-fold theLD₅₀ of a Botulinum neurotoxin (BoNT) and H7 and B5 VHH heterodimer withtwo E-tags and an anti-E-tag mAb (E/H7/B5/E+anti-E mAb) and a control(the toxin alone). Remarkably, 100% of the mice survived a 10,000 LD₅₀challenge of BoNT/A when administered the double-tagged heterodimer andthe anti-tag mAb.

FIG. 10 A-FIG. 10 B are nucleotide sequences and amino acid sequences ofrecombinant BoNT/A holotoxin binding VHHs: thioredoxin/JDQ-H7(H7)/E-tag(SEQ ID NO: 45), thioredoxin/JDQ-B5(B5)/E-tag (SEQ ID NO: 47),thioredoxin/H7/flexible spacer (fs)/B5/E-tag (SEQ ID NO: 49), andthioredoxin/E-tag/H7/fs/B5/E-tag (SEQ ID NO: 51). The correspondingamino acid sequences of the VHHs including amino acid sequences forthioredoxin/H7/E-tag (SEQ ID NO: 46), thioredoxin/B5/E-tag (SEQ ID NO:48), thioredoxin/H7/fs/B5/E-tag (SEQ ID NO: 50),thioredoxin/E-tag/H7/fs/B5/E-tag (SEQ ID NO: 52), and thioredoxin (SEQID NO: 53) are shown.

FIG. 11 A-FIG. 11 B are Meyer-Kaplan survival plots showing percentsurvival (% survival, ordinate) of subjects as a function of time indays (abscissa) following contact with BoNT/A and later time (1.5 hoursor three hours later) administered VHH binding/neutralizing agents.Subjects (five per group) were intravenously exposed to 10 LD₅₀(ten-fold LD₅₀) of BoNT/A, and then later administered either: a mixtureof 1 μg ciA-H7 monomer (SEQ ID NO: 32) and 1 μg of ciA-B5 monomer (SEQID NO: 24); H7/B5 heterodimeric protein (SEQ ID NO: 58); a sheepantitoxin serum; or control (no binding agent). Data show that the H7/B5heterodimer was effective as an antitoxin neutralizing agent andprotected subjects from the lethal challenge of BoNT/A.

FIG. 11 A shows percent survival for subjects exposed to ten-fold LD₅₀of BoNT/A then administered 1.5 hours later either a mixture of H7 andB5 monomers; H7/B5 heterodimer; a sheep serum antitoxin; or controltoxin only (no agents).

FIG. 11 B shows percent survival for subjects exposed to ten-fold LD₅₀of BoNT/A then administered three hours later either a mixture of H7 andB5 monomers; H7/B5 heterodimer; a sheep serum antitoxin; or controltoxin only (no agents).

FIG. 12 A-FIG. 12 C are line graphs showing that VHH monomers and VHHheterodimers neutralized C. difficile toxin b (TcdB) and protectedsubjects from death caused by exposure to TcdB.

FIG. 12 A is a line graph showing that VHH monomers neutralized C.difficile toxin B (TcdAB) and protected cells from the toxin. Thepercent CT26 cells affected by TcdB (% affected; ordinate) is shown as afunction of concentration (0.003 nM, 0.03 nM, 0.3 nM, 3 nM, 30 nM, 300nM, or 3000 nM) of administered VHH monomers: 5D (circle), 2D (square),or E3 (light upward facing triangle). Control cells were administeredtoxin only (TcdB; dark downward facing triangle). Strength ofneutralizing VHH activity was observed in the order 5D as strongestfollowed by E3 and 2D.

FIG. 12 B is a line graph showing percent of cells affected by TcdB (%affected; ordinate) as a function of concentration of administeredmixture of 5D and E3 monomers, 5D/E3 heterodimer (VHH; abscissa), or atoxin only control. It was observed that the 5D/E3 VHH heterodimer(squares) was about ten-fold more potent as toxin neutralizing agentthan the mixture of 5D monomer and E3 monomer (triangles).

FIG. 12 C is a Meyer-Kaplan survival plot of a C. difficile infectionmodel showing percent mouse survival (ordinate) as a function of time(hours post challenge, abscissa) of subjects co-administered toxin andVHH neutralizing agents. Subjects were co-administered a lethal dose ofTcdB with: a mixture of 10 μg of 5D monomers and E3 monomers (5 μg ofeach monomer per mouse; dashed line, blue); a mixture of 1 μg of 5Dmonomers and E3 monomers (500 ng of each monomer per mouse; thick solidline, blue), 5D/E3 heterodimer (250 ng per mouse; light solid line,red), or phosphate-buffered saline (PBS; thin solid line, black).Percent survival was calculated for each group of subjects.

FIG. 13 A-FIG. 13 C are amino acid sequences for VHH monomers and VHHheterodimers designed to specifically bind epitopes of botulism toxinsserotype A (BoNT/A) and serotype B (BoNT/B). Each VHH was purified fromE. coli as a thioredoxin fusion protein having a singlecarboxyl-terminal epitopic tag (tag or E-tag).

FIG. 13 A is a set of amino acid sequences of VHH monomers thatspecifically recognize and bind to epitopes on BoNT/A (ciA-A5, ciA-B5,ciA-D12, ciA-F12, ciA-G5, and ciA-H7) and epitopes of BoNT/B (ciB-A11,ciB-B5, ciB-B9, and ciB-H11). The sequences are aligned to showhomology. Dashed regions of the amino acid sequences are spaces insertedto align the amino acid regions.

FIG. 13 B is a set of amino acid sequences of VHH monomers (ciA-D1,ciA-H5, and ciA-H11) that bind specifically to the same epitope ofBoNT/B as ciA-H7.

FIG. 13 C shows amino acid sequences for double-tagged VHH heterodimers,ciA-H7/ciA-B5(2E) and ciA-F12/CiA-D12(2E), that specifically bindBoNT/A.

FIG. 14 A-FIG. 14 B are photographs of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analyses of VHH monomersand VHH heterodimers.

FIG. 14 A shows SDS-PAGE analysis of the tagged (E) VHH monomers ciA-D1,ciA-H4, ciA-H11, ciA-A5, ciA-C2, ciA-D12, ciA-F12, ciA-G5, and ciA-H7.

FIG. 14 B is a SDS-PAGE analysis of single- or double-tagged VHHheterodimers including: ciA-H7/ciA-B5 singly tagged on ciA-B5 (leftchannel); double tagged ciA-H7/ciA-B5 having a tag on both ciA/H7 andciA-B5 (second channel from left), ciA-F12/ciA-D12 singly tagged onciA-B5 (third channel from the left); double tagged ciA-F12/ciA-D12having a tag on both ciA/F12 and ciA-D12 (fourth channel from left),double tagged ciA-A11/ciA-B5 having a tag on both ciA/A11 and ciA-B5(right channel).

FIG. 15 are photographs of Western blots showing ability of VHH monomersto prevent BoNT/A from cleaving synaptosomal-associated protein 25(SNAP25) in primary neurons in culture.

FIG. 16 A-FIG. 16 C is a set of drawings and Meyer-Kaplan survival plotsshowing that mouse subjects administered each of a set of mixtures ofVHH monomers in combination with anti-tag clearing antibody wereprotected from BoNT/A.

FIG. 16 A (top) is a drawing of a BoNT/A bound to two different taggedbinding protein monomers that are each specifically bound by an anti-tagantibody. FIG. 16 A (bottom) is a set of graphs showing percent ofsurvival (% survival, ordinate) as a function of time (days, abscissa)of subjects co-administered 100-fold (FIG. 16 A bottom left graph) or1,000-fold (FIG. 16 A bottom right graph) the LD₅₀ of a BoNT/A andcombinations of VHH monomers (ciA-D12 and ciA-F12) with or withoutanti-tag clearing antibody (+αE and −αE respectively). The mixture ofVHH monomer B5, VHH monomer H7 and anti-tag clearing antibody protectedsubjects from the 100-fold LD50 of toxin.

FIG. 16 B (top) is a drawing of a BoNT/A bound to three differentmonomeric tagged binding protein each specifically bound by an anti-tagantibody. FIG. 16 B (bottom) is a set of graphs showing percent survivalon the ordinate as a function of time (days, abscissa) of subjectsco-administered 1,000-fold BoNT/A LD₅₀ (FIG. 16 B bottom left graph) or10,000-fold BoNT/A LD₅₀ (FIG. 16 bottom B right graph), and combinationsof three VHH monomers with or without anti-tag clearing antibody (+αEand −αE respectively).

FIG. 16 C (top) is a drawing of a BoNT/A bound to four different taggedbinding protein monomers that are each specifically bound by an anti-tagantibody. FIG. 16 C (bottom) is a set of graphs showing percentsurvival, ordinate, of subjects as a function of time (days, abscissa)of subjects co-administered 1,000-fold BoNT/A LD₅₀ (FIG. 16 C bottomleft graph) or 10,000-fold BoNT/A LD₅₀ (FIG. 16 C bottom right graph),and a mixture of ciA-B5, ciA-H7, ciA-D12 and ciA-F12 VHH monomers with(+αE) or without (−αE) anti-tag clearing antibody.

FIG. 17 are graphs showing percent survival, ordinate, of subjects as afunction of time (days, abscissa) of subjects co-administered 1,000-foldBoNT/A LD₅₀ (FIG. 17 left graph) or 10,000-fold BoNT/A LD₅₀ (FIG. 17right graph), and mixtures of VHH monomers and anti-tag clearingantibody (αE). Control subjects received toxin only. Unless indicatedotherwise, an asterisk (*) in FIGS. 17-24 indicates that the subjectsadministered the VHH monomer or multimer displayed no symptoms of toxinexposure.

FIG. 18 A-FIG. 18 B are a table showing affinity binding data for VHHsand a set of line graphs showing improved protection of subjects fromvery large doses of BoNT/A following administration of each of sets ofmixtures of VHH monomers with strong affinity for BoNT/A and clearingantibody.

FIG. 18 A is a table showing binding affinities (Kd) determined bysurface plasmon resonance (SPR) analysis of each of VHH monomers ciA-H7,ciA-D1, ciA-H4, and ciA-H11. SPR analysis was used to determine thebinding affinities to epitope A1 of BoNT/A for each VHH monomer. H7 hasthe greatest affinity and H11 the least affinity.

FIG. 18 B is a set of graphs showing percent survival on the ordinate ofsubjects as a function of time (days, abscissa) followingco-administration of BoNT/A at 100-fold (FIG. 18 B left graph) or1,000-fold (FIG. 18 B right graph) the LD₅₀, and a mixture of two VHHmonomers (B5+C2) or a mixture of three VHH monomers with anti-tagclearing antibody: B5+C2+H11; B5+C2+H7; B5+C2+D1; or B5+C2+H2.

FIG. 19 A-FIG. 19 B are drawings and graphs showing that administeringheterodimers composed of neutralizing VHH components resulted in greaterantitoxin efficacy than heterodimers composed of non-neutralizing VHHs,and that presence of two or more E-tags within the VHH heterodimersfurther increased the antitoxin efficacy.

FIG. 19 A (top) is a drawing of a BoNT/A bound to two different taggedheterodimer binding proteins that are each specifically bound by ananti-tag antibody. FIG. 19 A (bottom) is a set of graphs showing percentsurvival on the ordinate of subjects as a function of time (days,abscissa) after co-administration of 1,000-fold (FIG. 19 A bottom leftgraph) or 10,000-fold (FIG. 19 A bottom right graph) the BoNT/A LD₅₀,and a VHH heterodimer composition with (+αE) or without (−αE) anti-tagclearing antibody. The tagged VHH heterodimer composition was eithercomposed of neutralizing VHHs ciA-H7 and ciA-B5 (H7/B5), or ofnon-neutralizing VHHs ciA-D12 and ciA-F12 (D12/F12). Data show thatsubjects administered the heterodimer composition containingneutralizing VHHs ciA-B5 and ciA-H7 survived longer than subjectsadministered the heterodimer composition containing non-neutralizingVHHs ciA-D12 and ciA-F12. Subjects administered clearing anti-tagantibodies generally survived longer than subjects not administeredclearing-tag antibodies.

FIG. 19 B (top) is a drawing of a BoNT/A bound to two differentdouble-tagged heterodimer binding proteins that are each specificallybound by two anti-tag antibodies. FIG. 19 B (bottom) is a set of graphsshowing percent survival, ordinate, of subjects as a function of time(days, abscissa) after co-administration of an amount of BoNT/A1,000-fold (FIG. 19 B bottom left graph) or 10,000-fold (FIG. 19 Bbottom right graph) the LD₅₀, and double tagged VHH heterodimers with(+αE) or without (−αE) anti-tag clearing antibody. Subjects administeredneutralizing ciA-B5/ciA-H7 heterodimer survived longer than subjectsadministered non-neutralizing ciA-D12/ciA-F12 heterodimer. Data showthat all subjects administered double-tagged ciA-B5/ciA-H7 heterodimersand anti-tag clearing antibody survived exposure to 1,000-fold (FIG. 19B bottom left graph) or 10,000-fold the LD₅₀ of BoNT/A (FIG. 19 B bottomright graph).

FIG. 20 is a set of graphs showing percent survival on the ordinate ofsubjects as a function of time (days, abscissa) after co-administrationof 100-fold (FIG. 20 left graph) or 1,000-fold (FIG. 20 right graph)BoNT/A LD₅₀, and multi-tagged VHH heterodimers with anti-tag clearingantibody. The ciA-D12/ciA-F12 heterodimer protein contained either onetag (1e), two tags (2e), three tags (3e), or control no tag. Subjects(five mice per group) were administered 20 μg of the heterodimercomposition or the mixture of ciA-D12 and ciA-F12 monomers (20 μg ofeach monomer). Control subjects were administered neither monomer norheterodimer. Each subject received 60 picomoles of anti-E-tag clearingantibody. Data show that subjects administered ciA-D12/ciA-F12heterodimers having either one tag or two tags survived (100% survival)the challenge of 100-fold the LD₅₀ of BoNT/A (FIG. 20 left graph).Subjects receiving 1,000-fold the LD₅₀ of BoNT/A and ciA-D12/ciA-F12heterodimers with clearing antibody died within one day followingchallenge with independent of number of tags (FIG. 20 right graph).

FIG. 21 is a set of graphs showing percent survival, ordinate, ofsubjects treated with different amounts of anti-tag clearing antibody asa function of time (days, abscissa) after exposure to BoNT/A 100-fold(FIG. 21 left graph) or 1,000-fold (FIG. 21 right graph) the LD₅₀ and todouble tagged ciA-D12/ciA-F12 heterodimer (20 picomoles). Anti-tagclearing antibody was administered at: 20 picomoles, 40 picomoles, 60picomoles, 120 picomoles, or control (none). Control subjects receivedtoxin only (no agents). Data show improved antitoxin efficacy insubjects co-administered amounts (40, 60 or 120 picomoles) increasedanti-tag clearing antibody compared to 20 picomoles.

FIG. 22 is a graph showing percent survival, ordinate, of subjectstreated with different doses of double tagged neutralizing ciA-B5/ciA-H7heterodimers as a function of time (days, abscissa) for subjectsco-administered 1,000-fold BoNT/A LD₅₀, and anti-tag clearing antibody.Heterodimer ciA-B5/ciA-H7 was administered in doses of: 1.5 picomoles,4.4 picomoles, 13 picomoles, or 40 picomoles. Control subjects receivedtoxin only (no agents). Data show complete survival after seven days ofsubjects receiving amounts of 13 picomoles or 40 picomoles double taggedneutralizing ciA-B5/ciA-H7 heterodimer, such that than 13 picomolesprotected subjects fully from 1,000-fold BoNT/A LD₅₀, compared to 1.5picomoles or 4.4 picomoles (no survival after one day).

FIG. 23 A-FIG. 23 B are graphs showing percent survival, ordinate, aftersubjects were exposed to ten-fold BoNT/A LD₅₀ and were administereddouble-tagged heterodimer and anti-tag clearing antibody of subjects asa function of time (days, abscissa). Administration of heterodimer aftertoxin exposure was observed to have protected subjects from symptoms anddeath caused by exposure to ten-fold BoNT/A LD₅₀.

FIG. 23 A is a set of graphs showing percent survival of subjects as afunction administration of: double tagged ciA-D12/ciA-F12 heterodimerwith anti-tag clearing antibody (+αE), double tagged ciA-D12/ciA-F12heterodimer without anti-tag clearing antibody (−αE), a sheep serumantitoxin, or toxin only control (no agents). Prior to administration ofheterodimer, subjects were exposed 1.5 hours (FIG. 23 A left graph) orthree hours (FIG. 23 A right graph) to ten-fold BoNT/A LD₅₀. Data show100% survival of subjects administered ciA-D12/ciA-F12 heterodimer andanti-tag antibody after 1.5 hours. Survival of subjects administeredciA-D12/ciA-F12 heterodimer was comparable to that in subjectsadministered sheep serum antitoxin.

FIG. 23 B is a set of graphs showing percent survival of subjects as afunction administration of: double tagged ciA-B5 and ciA-H7 heterodimerwith anti-tag clearing antibody (+αE), or with double taggedciA-B5/ciA-H7 heterodimer without anti-tag clearing antibody (−αE), orwith a sheep serum antitoxin, or toxin only control (no agents). Priorto treatment with heterodimer, subjects were exposed to ten-fold BoNT/ALD₅₀ either 1.5 hours (FIG. 23 B left graph) or three hours (FIG. 23 Bright graph). Data show that subjects administered ciA-B5/ciA-H7heterodimer with or without anti-E tag antibody survived longer thansubjects administered sheep serum antitoxin. Survival of subjectsadministered ciA-B5/ciA-H7 heterodimer was greater than subjectsadministered sheep serum antitoxin.

FIG. 24 A-FIG. 24 B are line graphs showing that subjects administeredciA-A11/ciA-B5 heterodimers with anti-tag clearing antibody wereprotected from BoNT/B exposure.

FIG. 24 A is a graph showing survival on the ordinate as a function oftime (days, abscissa) co-administration of 1,000-fold (FIG. 24 A leftgraph) or 10,000-fold (FIG. 24 A right graph) BoNT/B LD₅₀ and acombination of ciB-A11 and ciB-B5 heterodimer with (+αE) or without(−αE) anti-tag clearing antibody, or toxin only control (no agents).Data show that subjects administered ciA-A11/ciA-B5 heterodimer andanti-E-tag clearing antibody survived and were protected longer fromBoNT/A than control subjects administered no agents and no anti-E tagantibody.

FIG. 24 B is a set of graphs showing subject survival (ordinate) as afunction of time, abscissa, after administration of: double taggedciB-A11 and ciB-B5 heterodimer and anti-tag clearing antibody (+αE), ordouble tagged ciB-A11 and ciB-B5 heterodimer without anti-tag clearingantibody (−αE), a sheep serum antitoxin, or toxin only control.Following 1.5 hours (FIG. 24 B left graph) or three hours (FIG. 24 Bright graph) exposure to ten-fold BoNT/B LD₅₀, the subjects wereadministered the heterodimer. A greater percentage of subjectsadministered ciB-A11 and ciB-B5 heterodimer survived exposure to BoNT/Bthan subjects administered sheep serum antitoxin.

FIG. 25 is a line graph of percent of cells affected by C. difficiletoxin A (TcdA) and protection of cells from the toxin by VHH monomers.The percent CT26 cells affected by TcdA (% affected; ordinate) is shownas a function of concentration (0.1 nM, 0.48 nM, 2.4 nM, 12 nM, 60 nM,or 300 nM) of each administered VHH monomer: A3H (circle), A11G (lightsquare); AC1 (upward dark empty triangle), AE1 (upward light triangle),AH3 (downward triangle), or AA6 (dark empty square). Control cells wereadministered toxin only (TcdA; dark downward triangle). Strength ofneutralizing VHH activity was observed in the following order: AA6 asstrongest, then AH3, AC1, AE1, A11G, and A3H as weakest.

FIG. 26 is a line graph showing percent CT26 cells affected after 24hours of TcdA exposure (ordinate) as a function of concentrationadministered (abscissa: 0.03 ng/mL, 0.1 ng/mL, 1 ng/mL, 3 ng/mL, 10ng/mL, 30 ng/mL, 100 ng/mL, 300 ng/mL, or 1000 ng/mL), or toxin onlycontrol (TcdA; vertical line). Agents administered were: VHH monomer AH3(AH3, diamond), VHH monomer AA6 (AA6, square), a mixture of VHH monomersAH3 and AA6 (AH3+AA6, triangle), VHH heterodimer of AH3 and AA6(AH3/AA6, −x−); or a homodimer of heterodimer (tetramer) containing AH3and AA6 using a dimerizer sequence oAgB (AH3/AA6/oAgB, stars; SEQ ID NO:95). Control cells were treated with medium only. Percent cell roundingwas analyzed using a phase contrast microscope. It was observed that thehomodimer of the heterodimer containing AH3 and AA6 resulted in thestrongest TcdA neutralization.

FIG. 27 is a set of line graphs showing percent affected CT26 cellsexposed to toxin (ordinate) and then contacted with VHH heterodimer of5D and AA6 (FIG. 27 top graph) or with heterodimer of 5D and AH3 (FIG.27 bottom graph) as a function of concentration of VHH (abscissa: 0.01nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, or 30 nM). CT26 cellswere exposed overnight to TcdA (2 ng/mL; diamond) or TcdB (0.1 ng/mL;square), and then treated with either heterodimer 5D/AA6 (FIG. 27 topgraph) or heterodimer 5D/AH3 (FIG. 27 bottom graph). Each heterodimerincluded a VHH monomer (5D) that neutralized TcdB, and a VHH monomer(AA6 or AH3) that neutralized TcdA. Data show that the treatment waseffective to protect cells from both toxins.

FIG. 28 A-FIG. 28 C are a drawing, a line graph and a bar graph showingthat a VHH heterodimer of 5D and AA6 protected mouse subjects from TcdAand TcdB in an oral C. difficile spore challenge model.

FIG. 28 A is a protocol for a clinically relevant murine C. difficileinfection model. Administration of VHH is given after a spore challenge.

FIG. 28 B shows percent survival (ordinate) as a function of timefollowing spore challenge (abscissa) for subjects administered 5D/AA6heterodimer as described in FIG. 28 A. Data show that after the sporechallenge, 90% of 5D/AA6 heterodimer contacted-subjects survived, andall control subjects not administered 5D/AA6 heterodimer or other agentdied within two days.

FIG. 28 C showing percent diarrhea (ordinate) as a function of timefollowing spore challenge (abscissa) for subjects administered 5D/AA6heterodimer (5D/AA6 TrxA; left bar), or control PBS (right bar) asdescribed in FIG. 28 A. Data show that 5D/AA6 heterodimeradministered-subjects were five-fold less likely to display symptoms ofdiarrhea than control untreated subjects.

FIG. 29 A-FIG. 29 B are amino acid sequences and nucleotide sequencesfor VHHs that specifically bind either Shiga toxin, anthrax protectiveantigen, ricin A chain (RTA) antigen, or ricin B chain (RTB) antigen.The nucleotide SEQ ID NOs: 131, 155 and 159 shown in FIG. 29 B includethe letter “N” in the nucleotide sequences in FIG. 29 B which indicatesa position in the nucleotide sequence for which an adenine (A) residueor a guanine (G) residue may be inserted to encode the correspondingamino acid in FIG. 29 A.

FIG. 29 A shows a list of amino acid sequences of VHHs identified thatbind each target as indicated:

Shiga toxin: JET-H12 (SEQ ID NO: 96) and JFG-H6 (SEQ ID NO: 98);

anthrax protective antigen: JHD-B6 (SEQ ID NO: 100), JHE-D9 (SEQ ID NO:102), JIJ-A12 (SEQ ID NO: 104), JIJ-B8 (SEQ ID NO: 106), JIJ-C11 (SEQ IDNO: 108), JIJ-D3 (SEQ ID NO: 110), JIJ-E9 (SEQ ID NO: 112), JIJ-F11 (SEQID NO: 114), JIK-B8 (SEQ ID NO: 116), JIK-B10 (SEQ ID NO: 118), JIK-B12(SEQ ID NO: 120), and JIK-F4 (SEQ ID NO: 122);

RTA: JIV-F5 (SEQ ID NO: 124), JIV-F6 (SEQ ID NO: 126), JIV-G12 (SEQ IDNO: 128), JIY-A7 (SEQ ID NO: 130), JIY-D9 (SEQ ID NO: 132), JIY-D10 (SEQID NO: 134), JIY-E1 (SEQ ID NO: 136), JIY-E3 (SEQ ID NO: 138), JIY-E5(SEQ ID NO: 140), JIY-F10 (SEQ ID NO: 142), and JIY-G11 (SEQ ID NO:144); and,

RTB: JIW-B1 (SEQ ID NO: 146), JIW-C12 (SEQ ID NO: 148), JIW-D12 (SEQ IDNO: 150), JIW-G5 (SEQ ID NO: 152), JIW-G10 (SEQ ID NO: 154), JIZ-B7 (SEQID NO: 156), JIZ-B9 (SEQ ID NO: 158), JIZ-D8 (SEQ ID NO: 160), andJIZ-G4 (SEQ ID NO: 162).

FIG. 29 B shows a list of nucleotide sequences that encode the VHH aminoacid sequences listed in FIG. 29 A. The nucleotide sequences encode VHHsthat bind each target as indicated:

Shiga toxin: JET-H12 (SEQ ID NO: 97) and JFG-H6 (SEQ ID NO: 99);

anthrax protective antigen: JHD-B6 (SEQ ID NO: 101), JHE-D9 (SEQ ID NO:103), JIJ-A12 (SEQ ID NO: 105), JIJ-B8 (SEQ ID NO: 107), JIJ-C11 (SEQ IDNO: 109), JIJ-D3 (SEQ ID NO: 111), JIJ-E9 (SEQ ID NO: 113), JIJ-F11 (SEQID NO: 115), JIK-B8 (SEQ ID NO: 117), JIK-B10 (SEQ ID NO: 119), JIK-B12(SEQ ID NO: 121), and JIK-F4 (SEQ ID NO: 123);

RTA: JIV-F5 (SEQ ID NO: 125), JIV-F6 (SEQ ID NO: 127), JIV-G12 (SEQ IDNO: 129), JIY-A7 (SEQ ID NO: 131), JIY-D9 (SEQ ID NO: 133), JIY-D10 (SEQID NO: 135), JIY-E1 (SEQ ID NO: 137), JIY-E3 (SEQ ID NO: 139), JIY-E5(SEQ ID NO: 141), JIY-F10 (SEQ ID NO: 143), and JIY-G11 (SEQ ID NO:145); and,

RTB: JIW-B1 (SEQ ID NO: 147), JIW-C12 (SEQ ID NO: 149), JIW-D12 (SEQ IDNO: 151), JIW-G5 (SEQ ID NO: 153), JIW-G10 (SEQ ID NO: 155), JIZ-B7 (SEQID NO: 157), JIZ-B9 (SEQ ID NO: 159), JIZ-D8 (SEQ ID NO: 161), andJIZ-G4 (SEQ ID NO: 163).

FIG. 30A-FIG. 30D are a set of line graphs showing VHH binding to Stx1toxin as a function of input VHH concentration. Dilution ELISAs wereperformed by coating plates with 0.5 μg/ml of 4D3 mAb Stx1. The plateswere blocked and then incubated with 0.3 μg/ml of Stx1. For standardELISAs, plates were coated with 1.5 μg of Stx1. VHH agents to be testedwere serially diluted, incubated for 1 hour at room temperature, washedand the bound VHH agent were detected with HRP-anti-E-tag. The boundVHH-HRP tagged agents were detected using the TMB kit by Sigma andvalues were plotted as a function of the input VHH concentration.

FIG. 30A is a line graph of Stx-A4 VHH, Stx-A5 VHH and heterodimerStxA4-A5 VHH binding to Stx1 toxin as a function of input VHHconcentration. VHH heterodimer Stx A4-A5 is displayed by dotted line.

FIG. 30B is a line graph of Stx-A4 VHH, Stx1-A9 VHH and heterodimerStxA4-A9 VHH binding to Stx1 toxin as a function of input VHHconcentration. VHH heterodimer Stx A4-A9 is displayed by dotted line.

FIG. 30C is a line graph of Stx1-A9 VHH, Stx1-D4 VHH and heterodimerStxA9-D4 VHH binding to Stx1 toxin as a function of input VHHconcentration. VHH heterodimer StxA9-D4 is displayed by dotted line.

FIG. 30D is a line graph of Stx1-A9 VHH, Stx-A5 VHH, Stx2-G1 VHH andheterotrimer StxA9-A5-G1 VHH binding to Stx1 toxin as a function ofinput VHH concentration. VHH heterotrimer StxA9-A5-G1 is displayed bydashed line.

FIG. 31A-FIG. 31D are a set of line graphs showing VHH binding to Stx1toxin as a function of input VHH concentration. Dilution ELISAs wereperformed by coating plates with 0.5 μg/ml of 3D1 mAb Stx2. The plateswere blocked and then incubated with 0.3 μg/ml of Stx1. For standardELISAs, plates were coated with 1.5 μg of Stx2. VHH agents to be testedwere serially diluted, incubated for 1 hour at room temperature, washedand the bound VHH agent were detected with HRP-anti-E-tag. The boundVHH-HRP tagged agents were detected using the TMB kit by Sigma andvalues were plotted as a function of the input VHH concentration.

FIG. 31A is a line graph of Stx-A4 VHH, Stx-A5 VHH and heterodimerStxA4-A5 VHH binding to Stx2 toxin as a function of input VHHconcentration. VHH heterodimer Stx A4-A5 is displayed by dotted line.

FIG. 31B is a line graph of Stx2-D10 VHH, Stx2-G1 VHH and heterodimerStx G1-D10 VHH binding to Stx2 toxin as a function of input VHHconcentration. VHH heterodimer Stx G1-D10 is displayed by dotted line.

FIG. 31C is a line graph of Stx-A5 VHH, Stx2-D10 VHH, heterodimerStx-A5-D10 VHH and heterotrimer Stx A9-A5-D10 VHH binding to Stx2 toxinas a function of input VHH concentration. VHH heterotrimer Stx A9-A5-D10is displayed by dashed line.

FIG. 31D is a line graph of Stx1-A9 VHH, Stx-A5VHH, Stx2-G1 VHH andheterotrimer StxA9-A5-G1 VHH binding to Stx2 toxin as a function ofinput VHH concentration. VHH heterotrimer StxA9-A5-G1 is displayed bydashed line.

FIG. 32A-FIG. 32B are schematic drawings showing binding of multipleefAb molecules to Shiga toxin directed by a double-tagged VHHheterodimer targeting two epitopes (called a VNA), or to a single-taggedVHH-monomer which binds the pentameric B subunit.

FIG. 32A is a schematic drawing of a VHH-heterodimer VNA binding to atoxin, such as Shiga toxin (Stx), at two separate, non-overlappingepitopes. If the heterodimer contains two copies of an epitopic ‘tag’,then two molecules of the anti-tag efAb bind each bound heterodimermolecule leading to decoration of each toxin molecule by four efAbmolecules.

FIG. 32B is a schematic drawing of a VHH-monomer binding to an epitopethat is present at multiple sites on the toxin, such as the pentamericB-subunit of Stx, thereby binding at multiple sites on the toxin. If theVHH contains an epitopic tag, the efAb decorates each toxin molecule atfive sites.

FIG. 33A-FIG. 33D are a set of line graphs of Stx 1 toxin neutralizationin a cell based assay as a function of VHH agent concentration. A Stx1dose (about 15 pmoles) that induced about 100% Vero cell and killed themafter 48 hours was selected. A VHH monomer, VHH monomer pool or VHHheterodimer, as labeled, were pre-mixed with Stx1 in culture medium andapplied to Vero cells. Toxin neutralization was assessed after 48 hoursby cell staining at A590 as described in examples herein. The extent ofcell staining was plotted as a function of the VHH-agent concentrationemployed.

FIG. 33A is a line graph of Stx 1 toxin neutralization in a cell basedassay by Stx-A4 VHH, Stx-A5 VHH, a monomer pool of Stx-A4 and Stx-A5VHHs and heterodimer Stx-A4-A5 VHH as a function of VHH concentration.VHH heterodimer Stx-A4-A5 is displayed by dotted line.

FIG. 33B is a line graph of Stx 1 toxin neutralization in a cell basedassay by Stx-A4 VHH, Stx1-A9 VHH, a monomer pool of Stx-A4 and Stx1-A9VHHs and heterodimer Stx1-A4-A9 VHH as a function of VHH concentration.VHH heterodimer Stx-A4-A9 is displayed by dotted line.

FIG. 33C is a line graph of Stx 1 toxin neutralization in a cell basedassay by Stx1-A9 VHH, Stx-D4 VHH, a monomer pool of Stx1-A9 and Stx-D4VHHs and heterodimer Stx1-A9-D4 VHH as a function of VHH concentration.VHH heterodimer Stx1-A9-D4 is displayed by dotted line.

FIG. 33D is a line graph of Stx 1 toxin neutralization in a cell-basedassay by Stx-A5 VHH, Stx1-A9 VHH, a monomer pool of Stx-A5 and Stx1-A5VHHs and heterotrimer Stx1-A9-A5-G1 VHH as a function of VHHconcentration. VHH heterotrimer Stx1-A9-A5-G1 is displayed by dashedline.

FIG. 34A-FIG. 34D are a set of line graphs of Stx 2 toxin neutralizationin a cell based assay as a function of VHH agent concentration. A Stx2dose (about 35 pmoles) that induced about 100% Vero cell and killed themafter 48 hours was selected. A VHH monomer, VHH monomer pool or VHHheterodimer, as labeled, were pre-mixed with Stx2 in culture medium andapplied to Vero cells. Toxin neutralization was assessed after 48 hoursby cell staining at A590 as described in examples herein. The extent ofcell staining was plotted as a function of the VHH-agent concentrationemployed.

FIG. 34A is a line graph of Stx 2 toxin neutralization in a cell basedassay by Stx-A4 VHH, Stx-A5 VHH, a monomer pool of Stx-A4 and Stx-A5VHHs and heterodimer Stx-A4-A5 VHH as a function of VHH concentration.VHH heterodimer Stx-A4-A5 is displayed by dotted line.

FIG. 34B is a line graph of Stx 2 toxin neutralization in a cell basedassay by Stx2-D10 VHH, Stx2-G1 VHH, a monomer pool of Stx2-D10 andStx2-G1VHHs and heterodimer Stx2-G1-D10 VHH as a function of VHHconcentration. VHH heterodimer Stx2-G1-D10 is displayed by dotted line.

FIG. 34C is a line graph of Stx 2 toxin neutralization in a cell basedassay by Stx-A5 VHH, Stx2-D10 VHH, a monomer pool of Stx-A5 and Stx2-D10VHHs, heterodimer Stx-A5-D10 VHH and heterotrimer Stx-A9-A5-D10 as afunction of VHH concentration. VHH heterodimer Stx1-A5-D10 is displayedby dotted line and VHH heterotrimer Stx-A9-A5-D10 is displayed by dashedline.

FIG. 34D is a line graph of Stx 2 toxin neutralization in a cell basedassay by Stx-A5 VHH, Stx2-G1 VHH, a monomer pool of Stx-A5 and Stx2-G1VHHs and heterotrimer Stx-A9-A5-G1 VHH as a function of VHHconcentration. VHH heterotrimer Stx-A9-A5-G1 is displayed by dashedline.

FIG. 35A-FIG. 35D are a set of Meyer-Kaplan survival plots for percentsurvival of subjects as a function of time in days following contactwith Stx1 toxin and later time administered VHH binding/neutralizingagents. Subjects, groups of five mice were injected with 20 pmoles ofStx1 premixed with 40 pmoles of the labeled VHH-based antitoxin agent(or 640 pmoles of VHH-A9 where indicated) and monitored for illness anddeath for one week. The percent survival is plotted as a function oftime. In some subjects, an 80 pmole dose of efAb was included in thetreatment.

FIG. 35A is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx1 toxin and then administered either Stx-A9 VHH,Stx-A9 (640 pm) VHH or no VHH agent.

FIG. 35B is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx1 toxin and then administered either Stx-A9 VHH,Stx-A4 VHH, Stx-A9-A4 heterodimer VHH or no VHH agent.

FIG. 35C is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx1 toxin and then administered either heterotrimerStx-A9-A5-D10 VHH without efAb, heterotrimer Stx-A9-A5-D10 VHH with efAbor no VHH agent.

FIG. 35D is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx1 toxin and then administered either heterotrimerStx-A9-A5-G1 VHH with efAb or no VHH agent.

FIG. 36A-FIG. 36D are a set of Meyer-Kaplan survival plots for percentsurvival of subjects as a function of time in days following contactwith Stx2 toxin and later time administered VHH binding/neutralizingagents. Subjects, groups of five mice were injected with 1 pmoles ofStx2 premixed with 40 pmoles of the labeled VHH-based antitoxin agentand monitored for illness and death for one week. The percent survivalis plotted as a function of time. In some subjects, an 80 pmole dose ofefAb was included in the treatment.

FIG. 36A is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx2 toxin and then administered either Stx-A5 VHH,Stx-D10 VHH, Stx-D10 with efAb, heterodimer Stx-A5-D10 VHH, or no VHHagent.

FIG. 36B is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx2 toxin and then administered either a mixture ofStx-A5 VHH and Stx-D10, Stx-A5-D10 heterodimer VHH or no VHH agent.

FIG. 36C is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx2 toxin and then administered either heterotrimerStx-A9-A5-D10 VHH without efAb, heterotrimer Stx-A9-A5-D10 VHH with efAbor no VHH agent.

FIG. 36D is a Meyer-Kaplan survival plot for percent survival ofsubjects exposed to Stx2 toxin and then administered either heterotrimerStx-A9-A5-G1 VHH with efAb, heterotrimer Stx-A9-A5-G1 VHH without efAbor no VHH agent.

FIG. 37A-FIG. 37D are a set of micrographs and a bar graph showing thatVNA plus efAb protect subjects from Stx2 induced renal damage.Formalin-fixed, paraffin embedded and hematoxylin and eosin stained 3 μmsections were examined by light microscopy from untreated age- andsex-matched controls (FIG. 37A), mice receiving the A9/A5/G1 VNA+efAb(FIG. 37B), and mice receiving only A9/A5/G1 VNA (FIG. 36C). The numbersof tubules with lesions such as epithelial apoptosis/necrosis,attenuation and restitution, hypertrophy, hyperplasia, luminal dilation,tubular atrophy/collapse, interstitial cell proliferation and earlyinterstitial fibrosis were quantified in 6 random 20× fields per mousetotaling 114 measurements and plotted in a bar graph (FIG. 37D).Examples of lesions are highlighted by black oval in FIG. 37B and theasterisks in FIG. 37C. (N.D.=None Detected)

FIG. 38 is a listing of amino acid sequences of VHHs selected forbinding to Stx1 or Stx2. Sequences shown begin within framework 1 At thesite of the primer binding employed in coding sequence DNA amplificationfrom the immune alpaca cDNA and continue through the end of framework 4.The parentheses at the end indicate whether the VHH contains a longhinge (lh) or a short hinge (sh). The three-complementarity determiningregions (CDRs) are indicated at the top.

FIG. 39 is a dendrogram of VHHs selected for binding to Stx1 or Stx2.The VHH sequences shown in FIG. 38 were analyzed for homology to createa dendrogram. Longer branch lengths indicate less sequence homology. Thecentral node labeled as the ‘cross-specific homology group’ indicateVHHs that recognize both Stx1 And Stx2 and possess significant homologyin CDR3 (see FIG. 38).

FIG. 40 is a photograph of Western blot for VHH binding to Stx1 AndStx2. Purified Stx1 and Stx2 were resolved by SDS-PAGE and the gelstained for protein (stain). Molecular weight markers are shown to theleft. Similar lanes containing Stx1 And Stx2 were transferred to filtersfor Western blot. The blots were incubated with 10 μg/ml of theindicated VHHs or control. Bound VHH was visualized with HRP/anti-E-tag.

FIG. 41 is the amino acid sequence of the full translation product ofthe exemplary protein referred to herein as VNA2-Tcd (SEQ ID NO: 170).The underlined portions of the sequence show four recombinant VHHbinding domains (see also SEQ ID NO: 174, SEQ ID NO: 164, SEQ ID NO:165, SEQ ID NO: 166). The sequence from residue number 184 to residenumber 318 is SEQ ID NO: 174. The sequence from residue number 343 toreside number 477 is SEQ ID NO: 164. The sequence from residue number505 to reside number 623 is SEQ ID NO: 165. The sequence from residuenumber 645 to reside number 773 is SEQ ID NO: 166. Residue numbers325-339, 483-497, and 627-641 include the flexible spacer amino acidsequence (GGGGS)₃ (SEQ ID NO: 55).

FIG. 42 is the coding nucleotide sequence of VNA2-Tcd (SEQ ID: 169).

FIG. 43 is the amino acid sequence of the full translation product ofmammalian cell secreted VNA2-Tcd (SEQ ID NO: 167).

FIG. 44 is a photograph of an SDS-PAGE gel showing the purified VNA2-Tcdheterotetramer produced in E. coli and purified by Ni-affinitychromatography followed by gel filtration chromatography. A total of 6ug, 3 ug, or 1.5 ug were loaded into the indicated lanes

FIG. 45 A-FIG. 45 D are a set of line graphs showing neutralization ofTcdA and TcdB by VNA-TcdA and VNA-TcdB respectively.

FIG. 45 A is a line graph showing that the VHH tetramer, VNA2-Tcd, bindsC. difficile toxin A (TcdA) with high affinity. The ELISA assay wasperformed by coating the microtiter plates with TcdA and incubation withVNA2-Tcd at concentrations of 0.008 nM, 0.04 nM, 0.2 nM, 1 nM, 5 nM, 25nM, or 125 nM, with visualization of binding using an HRP-conjugateddetection antibody.

FIG. 45 B is a line graph showing that the VHH tetramer, VNA2-Tcd, bindsC. difficile toxin B (TcdB) with high affinity. The ELISA assay wasperformed by coating the microtiter plates with TcdB and incubation withVNA2-Tcd at concentrations of 0.008 nM, 0.04 nM, 0.2 nM, 1 nM, 5 nM, 25nM, or 125 nM, with visualization of binding using an HRP-conjugateddetection antibody.

FIG. 45 C is a line graph showing that the VHH tetramer, VNA2-Tcd,neutralized C. difficile toxin A (TcdA) and protected cells from thetoxin. The percent of CT26 cells affected by TcdA (% affected, ordinate)is shown as a function of concentration (from 0.13 pM, 0.62 pM, 3.2 pM,16 pM, 80 pM, 400 pM, 2000 pM, and 10000 pM). Percent cell rounding wasanalyzed using a phase contrast microscope.

FIG. 45 D is a line graph showing that the VHH tetramer, VNA2-Tcd,neutralized C. difficile toxin B (TcdB) and protected cells from thetoxin. The percent of CT26 cells affected by TcdB (% affected, ordinate)is shown as a function of concentration (from 0.13 pM, 0.62 pM, 3.2 pM,16 pM, 80 pM, 400 pM, 2000 pM, and 10000 pM). Percent cell rounding wasanalyzed using a phase contrast microscope.

FIG. 46 is a Meyer-Kaplan survival plot showing percent survival (%survival, ordinate) of subjects as a function of time in days (abscissa)following contact with C. difficile toxin. Six-week-old female C57BL/6mice were treated via IP injection with 50 ug/mouse of purified VNA2-Tcdone hour prior to IP challenge with 100 ng/mouse of C. difficile toxin A(TcdA) and 200 ng/mouse of C. difficile toxin B (TcdB). Control micechallenged with TcdA and TcdB all died or became moribund within 4 hourspost challenge. Untreated, VNA2-Tcd alone, TcdA+VNA2-Tcd, TcdB+VNA2-Tcd,and TcdA/B+VNA2-Tcd treated animals showed no signs of systemic effectsand survived until study termination at 7 days post challenge.

FIG. 47A-FIG. 47 C are graphs showing neutralization of C. difficileinfection by VNA-TcD.

FIG. 47 A is a line graph showing results of the C. difficile infectionmouse study. Mice were treated with an antibiotic in the drinking waterfor 3 days, then received an IP injection of clindamycin. The next day,the animals were infected with 10⁶ C. difficile UK6 spores. Cohorts ofanimals were treated with 3 doses of VNA2-Tcd (2.5 mg/kg at 4, 24, and48 hrs after infection) or PBS. PBS-treated control animals displayedrapid weight loss while the VNA2-Tcd-treated animals displayed little orno weight loss.

FIG. 47 B is a bar graph showing results of the C. difficile infectionmouse study. Mice were treated with an antibiotic in the drinking waterfor 3 days, then received an IP injection of clindamycin. The next day,the animals were infected with 10⁶ C. difficile UK6 spores. Cohorts ofanimals were treated with 3 doses of VNA2-Tcd (2.5 mg/kg at 4, 24, and48 hrs after infection) or PBS. All of the PBS-treated control animalsdisplayed diarrhea for at least 3 days post infection, while only 20% ofVNA2-Tcd-treated animals had diarrhea on Day 1 which resolved by Day 2.

FIG. 47 C is a Kaplan-Meier line graph showing results of the C.difficile infection mouse study. Mice were treated with an antibiotic inthe drinking water for 3 days, then received an IP injection ofclindamycin. The next day, the animals were infected with 10⁶ C.difficile UK6 spores. Cohorts of animals were treated with 3 doses ofVNA2-Tcd (2.5 mg/kg at 4, 24, and 48 hrs after infection) or PBS.PBS-treated control animals rapidly became moribund and 60% of theanimals were dead by Day 3. In contrast, 100% of the VNA2-Tcd-treatedanimals survived.

FIG. 48 A-FIG. 48 B are a set of photographs showing descending colonsof C. difficile-infected control pigs.

FIG. 48 A is a photograph of the descending colon of a C.difficile-infected control pig treated with PBS. The colons displayedsevere dilatation, mesocolonic edema, multifocal hemorrhages, andthickening of the intestinal wall.

FIG. 48 B is a photograph of the descending colon of a C.difficile-infected control pig treated with VNA2-Tcd. The colons showminimal or no dilatation, no intestinal wall thickening or hemorrhages.

FIG. 49 A-FIG. 49 C are photographs and bar graph showing neutrophilfoci in the lamina of C. difficile infected piglets and histologysections of the intestinal lumen of C. difficile infected piglets.

FIG. 49 A is a bar graph that compares the amount of neutrophil foci inthe lamina of C. difficile infected piglets treated with VNA2-Tcd or PBS(Control). The VNA2-Tcd treated animals had significantly fewerneutrophil foci than the control animals.

FIG. 49 B is a photograph of a histology section of the intestinal lumenof C. difficile infected piglets treated with PBS (Control). Mucosalulceration, hemorrhage, and marked neutrophilic infiltration, eruptionof neutrophils and sloughed mucosa is apparent.

FIG. 49 C is a photograph of a histology section of the intestinal lumenof C. difficile infected piglets treated with VNA2-Tcd. Mild mucosalerosion and mild neutrophilic infiltration is observed.

FIG. 50 is an SDS-PAGE gel. The top panel represents an SDS-PAGE gel inwhich protein is stained with Coomassie. The bottom panel is a Westernblot of the same samples as in top panel, identifying proteins with anE-tag peptide, facilitating identification of the degradation products.The data shows that a heterotetrameric VNA (VNA2-Tcd) containing fourVHHs is rapidly cleaved to the monomeric VHH components of the VNA. Twodimeric VNAs, VNA-TcdA and VNA-TcdB, that each contain two VHHs alsopresent in VNA2-Tcd is more stable to GI enzyme digestion. The 50 kDafull-size protein is rapidly digested to a protein of ˜30 KDa thatcontains the VHH heterodimers.

FIG. 51 A-FIG. 51 B are a set of line graphs showing affinity ofVNA-TcdA and VNA-TcdB to C. difficile toxin A and toxin B respectively.

FIG. 51 A is a line graph showing that the VHH dimer, VNA-TcdA, lackingthe protease-sensitive sites, binds C. difficile toxin A (TcdA) with thesame affinity as the tetramer, VNA2-Tcd. VNA-TcdB did not show affinityfor TcdA. The ELISA assay was performed by coating the microtiter plateswith TcdA and incubation with VNA-TcdA, VNA-TcdB, or VNA2-Tcd atconcentrations of 0.008 nM, 0.04 nM, 0.2 nM, 1 nM, 5 nM, 25 nM, or 125nM, with visualization of binding using an HRP-conjugated detectionantibody.

FIG. 51 B is a line graph showing that the VHH dimer, VNA-TcdB, lackingthe protease-sensitive sites, binds C. difficile toxin B (TcdB) with thesame affinity as the tetramer, VNA2-Tcd. VNA-TcdA did not show affinityfor TcdB. The ELISA assay was performed by coating the microtiter plateswith TcdA and incubation with VNA-TcdA, VNA-TcdB, or VNA2-Tcd atconcentrations of 0.008 nM, 0.04 nM, 0.2 nM, 1 nM, 5 nM, 25 nM, or 125nM, with visualization of binding using an HRP-conjugated detectionantibody.

DETAILED DESCRIPTION

The presence of toxins in the circulation is the cause of a wide varietyof human and animal illnesses. Antitoxins are therapeutic agents thatprevent toxin infection or reduce further development of negativesymptoms in patients that have been exposed to a toxin (a processreferred to as “intoxication”). Typically, antitoxins are antiseraobtained from large animals (e.g., sheep, horse, and pig) that wereimmunized with inactivated or non-functional toxin. More recently,antitoxin therapies have been developed using combinations of antitoxinmonoclonal antibodies including yeast-displayed single-chain variablefragment antibodies generated from vaccinated humans or mice. SeeNowakowski et al. 2002. Proc Natl Acad Sci USA 99: 11346-11350;Mukherjee et al. 2002. Infect Immun 70: 612-619; Mohamed et al. 2005Infect Immun 73: 795-802; Walker, K. 2010 Interscience Conference onAntimicrobial Agents and Chemotherapy—50th Annual Meeting—Research onPromising New Agents: Part 1. Drugs 13: 743-745. Antisera and monoclonalantibodies can be difficult to produce economically at scale, usuallyrequiring long development times and resulting in problematic qualitycontrol, shelf-life and safety issues. New therapeutic strategies todevelop and prepare antitoxins are needed.

Antitoxins function through two key mechanisms neutralization of toxinfunction and clearance of the toxin from the body. Toxin neutralizationoccurs through biochemical processes including inhibition of enzymaticactivity and prevention of binding to cellular receptors. Antibodymediated serum clearance occurs subsequent to the binding of multipleantibodies to the target antigen (Daeron M. 1997 Annu Rev Immunol 15:203-234; Davies et al. 2002 Arthritis Rheum 46: 1028-1038; Johansson etal. 1996 Hepatology 24: 169-175; and Lovdal et al. 2000 J Cell Sci 113(Pt 18): 3255-3266). Multimeric antibody decoration of the target isnecessary to permit binding to low affinity Fc receptors (Davies et al.2002 Arthritis Rheum 46: 1028-1038 and Lovdal et al. 2000 J Cell Sci 113(Pt 18): 3255-3266). Without being limited by any particular theory ormechanism of action, it is here envisioned that an ideal antitoxintherapeutic would both promote toxin neutralization to immediately blockfurther toxin activity and also accelerate toxin clearance to eliminatefuture pathology if neutralization becomes reversed.

Effective clearance of botulinum neurotoxin (BoNT), a National Instituteof Allergy and Infectious Diseases (NIAID) Category A priority pathogen,is believed by some researchers to require three or more antibodiesbound to the toxin. Nowakowski et al. 2002. (Proc Natl Acad Sci USA 99:11346-11350) determined that effective protection of mice against highdose challenge of BoNT serotype A (BoNT/A) required co-administration ofthree antitoxin monoclonal antibodies, and that all three antibodiespresumably promoted clearance. Data have shown that administration of apool of three or more small binding agents, each produced with a commonepitopic tag, reduced serum levels of a toxin when co-administered withan anti-tag monoclonal antibody (Shoemaker et al. U.S. publishedapplication 2010/0278830 A1 published Nov. 4, 2010 and Sepulveda et al.2009 Infect Immun 78: 756-763, each of which is incorporated herein inits entirety). The tagged binding agents directed the binding ofanti-tag monoclonal antibody to multiple sites on the toxin, thusindirectly decorating the toxin with antibody Fc domains and leading toits clearance through the liver.

Pools of scFv domain binding agents with specificity for BoNT/A and eachcontaining a common epitopic tag (E-tag), had been shown to be effectivefor decorating the botulinum toxin with multiple anti-tag antibodies(Shoemaker et al. U.S. utility patent publication number 2010/0278830published Nov. 4, 2010 and U.S. continuation-in-part patent publicationnumber 2011/0129474 published Jun. 2, 2011, each of which isincorporated herein by reference in its entirety). Data showed that theadministration of binding agents and clearance antibodies to subjectsresulted in clearance via the liver with an efficacy in mouse assaysequivalent to conventional polyclonal antitoxin sera. Ibid. andSepulveda et al. 2009 Infect Immun 78: 756-763. The tagged scFvs toxintargeting agents and the anti-tag monoclonal antibodies were effectivefor treating subjects at risk for or having been contacted with adisease agent.

The use of small binding agents to direct the decoration of toxin withantibody permits new strategies for the development of agents withimproved therapeutic and commercial properties. Examples herein showthat a single recombinant heterodimeric binding protein/agent includingtwo or more high-affinity BoNT binding agents (camelid heavy-chain-onlyAb VH (VHH) domains) and two epitopic tags, co-administered with ananti-tag mAb, protected subjects from botulism caused negative symptomsand lethality. Further the binding protein resulted in antitoxinefficacy equivalent to and greater than conventional BoNT antitoxinserum in two different in vivo assays. Examples herein compareneutralizing or non-neutralizing binding agents administered with orwithout clearing antibody, and show the relative contributions of toxinneutralization and toxin clearance to antitoxin efficacy. Examplesherein show that both toxin neutralization and toxin clearancecontribute significantly to antitoxin efficacy in subjects. Toxinneutralization or toxin clearance using heterodimer binding proteinantitoxins sufficiently protected subjects from BoNT lethality in atherapeutically relevant, post-intoxication assay. Methods in Examplesherein optionally further include a clearing antibody for example amonoclonal anti-E-tag antibody.

It was observed in Examples herein that VHH binding agents thatneutralized toxin function significantly improved the antitoxin efficacyand even obviated the need for clearing antibody in a clinicallyrelevant post-intoxication BoNT/A assay. The methods, compositions andkits using the multimeric binding proteins described herein havewidespread application in antitoxin development and other therapies inwhich neutralization and/or accelerated clearance of a target moleculebenefits a patient. For example, the target molecule is an exogenousdisease agent that infects or is at risk to infect a patient. Exogenousdisease agent for example is a virus, a cancer cell, a fungus, abacterium, a parasite and a product thereof such as a pathogenicmolecule, a protein, a lipopolysaccharide, or a toxin. Alternatively,the molecule is an endogenous (body produced) molecule that is producedin the patient and that causes or produces harmful effects on thepatient. For example, the molecule is a hormone or a protein that isassociated with a disease or condition, e.g., inflammation, cancer,transplant rejection, kidney failure, or a defect in blood clotting suchas hemophilia and thrombophilia. In various embodiments, the diseaseagent is a toxin of C. difficile.

C. difficile is a gram-positive, spore forming, anaerobic bacterium thatis the leading cause of antibiotic-associated diarrhea, the severity ofwhich ranges from mild diarrhea to life threatening pseudomembranouscolitis (Bartlett J G. 200 2 N Engl J Med 346:334-9 and Feng et al.PCT/US10/58701 filed Dec. 2, 2010, each of which is incorporated byreference in its entirety). Pathogenic C. difficile strains excreteexotoxins A (TcdA) and B (TcdB) that have been intimately linked to itspathogenicity. Both TcdA and TcdB are enterotoxic, capable of inducingintestinal epithelial damage and increasing mucosal permeability, andhence are thought to be responsible for the pathogenesis of C.difficile-associated colitis (Kelly C P et al. 1998 Annu Rev Med49:375-90). C. difficile has emerged as a leading cause ofhospital-acquired enteric infections with rapidly escalating annualhealth care costs in the United States (Kyne L et al. 2002 Clin InfectDis 34:346-353). The severity of C. difficile-associated infectionsranges from mild diarrhea to life threatening pseudomembranous colitis(Bartlett J G et al. 2002 N Engl J Med 346:334-339; Borriello S P 1998Antimicrob Chemother 41 Suppl C:13-19). Several hospital outbreaks of C.difficile-associated diarrhea (CDAD), with high morbidity and mortalityin the past few years in North America, have been attributed to thewidespread use of broad-spectrum antibiotics.

The emergence of more virulent C. difficile strains contributes also tothe increased incidence and severity of the disease (Loo V G et al. 2005N Engl J Med 353:2442-2449; McDonald L C et al. 2005 N Engl J Med353:2433-2441). Antibiotic usage results in a reduction of commensalmicroflora in the gut, which permits C. difficile to proliferate moreextensively, leading to the further production of toxins (Owens J R etal. 2008 Clinical Infectious Diseases 46(s1):S19-531). C. difficileinfection (CDI) includes a range of symptoms varying from mild diarrheato severe fulminate lethal disease (Kuijper E J et al. 2007 Curr OpinInfect Dis 20(4):376-383). Recent outbreaks of highly virulent C.difficile strains (McDonald L C et al. 2005 N Engl J Med353(23):2433-2441; Loo V G et al. 2005 N Engl J Med 353(23):2442-2449)have increased the urgency to devote greater resources towards theunderstanding of the molecular, genetic, and biochemical basis for thepathogenesis, with a view to use such information to develop novelpreventive and treatment modalities.

A cell-based immunocytotoxicity assay for detecting C. difficile toxinsdescribed in Feng et al. (PCT/US2009/003055 published Nov. 19, 2009 asWO 2009/139919) uses an anti-C. difficile toxin A (TcdA) monoclonalantibody, named A1H3, which substantially enhanced the activity of TcdAon Fc gamma receptor I (FcγRI)-expressing cells (He X, Sun X, Wang J, etal. Antibody-enhanced, Fc{gamma}R-mediated endocytosis of C. difficiletoxin A. Infect Immun 2009). Feng et al. shows use of A1H3 enhancingantibody, in combination with an electronic sensing system to develop areal-time and ultrasensitive assay for the detection of biologicalactivity of C. difficile toxins.

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factorscontributing to pathogenic C. difficile strains. These strains areenterotoxic, inducing intestinal epithelial cell damage, disruptingepithelium tight junctions leading to increased mucosal permeability(Pothoulakis C et al. 2001 Am J Physiol Gastrointest Liver Physiol280:G178-183; Riegler M et al. 1995 J Clin Invest 95:2004-2011; SavidgeT C et al. 2003 Gastroenterology 125:413-420). Moreover, these toxinsinduce production of immune mediators, leading to subsequent neutrophilinfiltration and severe colitis (Kelly C P et al. 1994 J Clin Invest93:1257-1265; Kelly C P et al. 1998 Annu Rev Med 49:375-390). TcdA andTcdB are structurally homologous, and contain a putative N-terminalglucosyltransferase and a cysteine proteinase domain, a transmembranedomain, and a C-terminal receptor binding domain (von Eichel-Streiber Cet al. 1996 Trends Microbiol 4:375-382) (Dank T et al. 2008 Trends inmicrobiology 16:222-229; Voth D E et al. 2005 Clin Microbiol Rev18:247-263).

Interaction between the toxin C-terminus and the host cell receptorsinitiates a receptor-mediated endocytosis (Florin I et al. 1983 BiochimBiophys Acta 763:383-392; Karlsson K A 1995 Curr Opin Struct Biol5:622-635; Tucker K D et al. 1991 Infect Immun 59:73-78). Although theintracellular mode of action remains unclear, it has been proposed thatthe toxins undergo conformational change at low pH in the endosomalcompartment, leading to membrane insertion and channel formation (FlorinI et al. 1986 Microb Pathog 1:373-385; Giesemann T et al. 2006 J BiolChem 281:10808-10815; Henriques B et al. 1987 Microb Pathog 2:455-463;Qa'Dan M et al. 2000 Infect Immun 68:2470-2474). A host cofactor is thenrequired to trigger a second structural change which is accompanied byan immediate autocatalytic cleavage and release of theglucosyltransferase domain into cytosol (Pfeifer G et al. 2003 J BiolChem 278:44535-44541; Reineke J e al. 2007 Nature 446:415-419; Rupnik Met al. 2005 Microbiology 151:199-208). Once the glucosyltransferasedomain reaches the cytosol, it inactivates proteins of the Rho/Racfamily, leading to alterations of cytoskeleton and ultimately cell death(Just I et al. 1995 Nature 375:500-503; Sehr P et al. 1998 Biochemistry37:5296-5304).

The clinical manifestation of CDI is highly variable, from asymptomaticcarriage, to mild self-limiting diarrhea, to the more severepseudomembranous colitis. The prevalence of systemic complication anddeath in CDI has become increasingly common (Siemann M et al. 2000Intensive care medicine 26:416-421). In life-threatening cases of CDI,systemic complications are observed, including cardiopulmonary arrest(Johnson S et al. 2001 Annals of internal medicine 135:434-438), acuterespiratory distress syndrome (Jacob S S et al. 2004 Heart Lung33:265-268), multiple organ failure (Dobson G et al. 2003 Intensive caremedicine 29:1030), renal failure (Cunney R J et al. 1998 Nephrol DialTransplant 13:2842-2846), and liver damage (Sakurai T et al. 2001 JInfect Dis 33:69-70). The exact reason for these negative complicationsis unclear, and may be caused by entry of the toxin into the circulationand systemic dissemination (Hamm E E et al. 2006 Proc Natl Acad Sci USA103:14176-14181).

Standard therapy depends on treatment with vancomycin or metronidazole,neither of which is fully effective (Zar et al. 2007 Clinical InfectiousDiseases 45:302-307). Moreover, an estimated 15% to 35% of thoseinfected with C. difficile relapse following treatment (Barbut et al.2000 J Clin Microbiol 38: 2386-2388; Tonna et al: Postgrad Med J 81:367). Unfortunately, the primary treatment option for recurrent CDI isstill metronidazole or vancomycin. Other options, such as probiotics,toxin-absorbing polymer and anion-exchange resins, have limited efficacy(Gerding, D. N., Muto, C. A. & Owens, R. C., Jr. 2008 Clin Infect Dis 46Suppl 1: S32-42). Therefore, immune-based therapies are a promisingapproach to control the disease. Antibodies specific for both of thesetoxins, and not against TcdA or TcdB alone, protect against toxigenic C.difficile infection in a hamster model (Libby et al, 1982 Infect Immun36: 822-829; Fernie et al, 1983 Dev Biol Stand 53: 325; and Kim et al,2006 Infection and immunity 74: 6339). Human serum antibodies specificfor both TcdA and TcdB are associated also with protection againstsymptomatic disease and recurrence. Recent phase II clinical trial ledby Merck demonstrated that the systemically administered human IgGmonoclonal antibodies against TcdA and TcdB prevents disease relapse inCDI patients (Lowy et al, 2010 The New England journal of medicine 362:197). However, the treatment involved the injection of a large quantityof two individual antibodies against each toxin.

Examples herein show, among others, a new approach to the development ofantitoxins that employs a single recombinant protein to promote toxindecoration with multiple copies of a single monoclonal antibody leadingto its neutralization and clearance from the body. The methods,compositions, and kits herein are useful for treating a great number ofthe most common pathogenic biological targets by acceleratingneutralization and clearance from the subject or patient.

Examples herein show, among others, that camelid VHH binding domains,which have multiple commercial advantages over scFvs due in part to theease and reduced cost of producing VHHs, were effective as toxintargeting agents both with and without being administered with clearingantibody. An important advantage of VHHs is the ability of medicalprofessionals and scientists to express these binding agents asheterodimers in which each component VHH remains fully functional. Themultimeric fusion proteins containing at least two VHH binding regionsresulted in the component VHHs binding to different epitopes on the sametoxin target. Without being limited by any particular theory ormechanism of action, it is believed that incorporation of two epitopetags on the heterodimers resulted in decoration of the toxin with twoclearing antibodies at each epitope, and resulted in a total of fourmonoclonal clearing antibodies binding to the heterodimers on the toxin.In addition, with certain heterodimers the decoration promoted efficienttoxin clearance. Either neutralization or clearance or both areimportant mechanisms of remediating toxin exposure. As eachdouble-tagged heterodimeric binding agent was bound only to only twomonoclonal antibodies, the heterodimeric agent itself may not beeffectively cleared by low affinity Fc receptors unless actually boundto the toxin.

The ability of antitoxin antibodies to protect mammalian subjects fromthe symptoms of toxin exposure is influenced by several factors that aredescribed herein. Examples herein used intoxication models and variedthe dose of antitoxin agent and the timing of antitoxin administrationrelative to exposure to toxin in order to determine whether both thedose and the timing of the antitoxin are factors that influenceantitoxin efficacy. In addition, examples herein analyzed the role thataffinity of the antibody for the toxin has on the ability of theantibody to bind (K_(on)) and remain bound (K_(off)) to the toxin andexert its effect. Data show that the ability of the antibodymonomer/heterodimer to inhibit the enzymatic activity of the toxinand/or prevent its entry into target cells (i.e. neutralization) is amajor factor in effective antitoxin treatment of subjects. Specifically,data show that the greater the binding affinity of the binding proteinto the target molecule, the greater the potential neutralization andclearance of the binding protein. Examples herein show also that themultimeric binding proteins promoted the clearance of the toxin from theserum and minimized further negative symptoms or lethality by the targetmolecule or disease agent. A portion of this work was published Jan. 6,2012 in the Public Library of Science One and was entitled, “A NovelStrategy for Development of Recombinant Antitoxin Therapeutics Tested ina Mouse Botulism”, authored by Jean Mukherjee, Jacqueline M. Tremblay,Clinton E. Leysath, Kwasi Ofori, Karen Baldwin, Xiaochuan Feng, DanielaBedenice, Robert P. Webb, Patrick M. Wright, Leonard A. Smith, SaulTzipori, and Charles B. Shoemaker (Mukherjee J. et al. 2012 PLoS One.7(1):e29941), which is incorporated by reference herein in its entirety.

Methods for engineering and selecting proteins for binding to diseaseagents are shown for example in U.S. utility application Ser. No.13/566,524 filed Aug. 3, 2012; U.S. publication number 2011/0129474published Jun. 2, 2011 (U.S. application Ser. No. 12/889,511 filed Sep.24, 2010), which is a continuation-in-part application of U.S.publication number 2010/0278830 published Nov. 4, 2010 (U.S. utilityapplication Ser. No. 12/032,744 filed Feb. 18, 2008), each of which isincorporated by reference herein in its entirety.

An aspect of the invention provides a method for treating a subject atrisk for exposure to or exposed to a disease agent, the methodincluding: contacting the subject with at least one recombinantheteromultimeric neutralizing binding protein including two or multiplebinding regions, such that the binding regions are not identical, andeach binding region specifically binds a non-overlapping portion of thedisease agent, such that the binding protein neutralizes the diseaseagent, thereby treating the subject for exposure to the disease agent.

In various embodiments of the method, the binding protein includes atleast one tag. For example, the tag is a molecule or epitope that isattached or genetically fused to the binding protein and/or bindingregions. The tag in various embodiments of the method inducesendogeneous clearance of the disease agent from the body in vivo. Forexample the tag includes SEQ ID NO: 15, or a variant thereof. In arelated embodiment, the tag includes an antibody epitope.

In certain embodiments of the method, the binding protein is selectedfrom: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Sac7d derivative (affitins, e.g. NANOFITINS, see Journal of MolecularBiology 2008 Nov. 28; 383(5):1058-68, the contents of which are herebyincorporated by reference), a Fv; a Fab; a Fab′; and a F(ab′)₂. In anembodiment, the binding protein is heterodimeric, for example thebinding protein has greater potency than each individual monomer. Inalternative embodiments, the heteromultimeric neutralizing bindingprotein is multimeric and the multimeric components are associatednon-covalently or covalently.

The binding protein in certain embodiments of the method includes alinker that separates multimeric components of the binding regions. Invarious embodiments, the linker includes at least one selected from: apeptide, a protein, a sugar, or a nucleotide. For example, the linkerincludes amino acid sequence GGGGS (SEQ ID NO: 54), or a variantthereof, or includes amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:55), or a variant thereof or a portion thereof. In a related embodiment,the linker is a flexible linker located within subunits/domains of thebinding protein, such that the linker does not negatively affect thefunction of the binding protein to the disease agent. For example, thelinker includes amino acid sequences/residues including serine andglycine, and in various embodiments is at least about three to fiveamino acids long, or about five to eight amino acids long, or abouteight to fifteen amino acids long.

In certain embodiments, the disease agent is a biological target orbiological molecule. For example, the biological target or thebiological molecule is naturally occurring within the subject, forexample a molecule or compound synthesized by the subject. An example ofa biological molecule synthesized by the subject is an IgE that isassociated with an allergy or an auto antibody or an MHC protein (e.g.,HLA class I antigens A and B and HLA class II antigen DR) associatedwith an autoimmune disease. For example the autoimmune disease isselected from: lupus erythematosus, Graves' disease, rheumatoidarthritis, Sjögren's syndrome, myasthenia gravis, and Hashimoto'sthyroiditis.

The disease agent in various embodiments of the method includes aplurality of non-identical disease agents, for example two or morebacterial toxins, or a viral toxin and a fungal species. In variousembodiments, the binding regions of the binding protein are specific toeach non-identical disease agent and bind to and neutralize theplurality of disease agents.

In various embodiments of the method, the disease agent is at least oneselected from: a virus, a cancer cell, a fungus, a bacterium, a parasiteand a product thereof such as a pathogenic molecule, a protein, alipopolysaccharide, and a toxin. In certain embodiments, the toxinincludes a protein, a lipid, a lipopolysaccharide, and a small moleculetoxin such as an aflatoxin or a dinoflagellate toxin. The toxin forexample is a Botulinum neurotoxin comprising a serotype selected from:A, B, C, D, E, F, and G. In certain embodiments of the method, the toxinis a Clostridium exotoxin comprising toxin A (TcdA) and toxin B (TcdB).

In various embodiments of the method, the toxin is at least one selectedfrom: staphylococcal α-hemolysin, staphylococcal leukocidin, aerolysincytotoxic enterotoxin, a cholera toxin, Bacillus cereus hemolysis IItoxin, a Helicobacter pylori vacuolating toxin, a Bacillus anthracistoxin, a cholera toxin, a Escherichia coli serotype O157:H7 toxin, aEscherichia coli serotype 0104:H7 toxin, a lipopolysaccharide endotoxin,a Shiga toxin, a pertussis toxin, a Clostridium perfringens iota toxin,a Clostridium spiroforme toxin, a Clostridium difficile toxin A, aClostridium difficile toxin B, a Clostridium septicum a toxin, and aClostridium botulinum C2 toxin. In a related embodiment of the method,the disease agent is an infectious strain, for example a bacterialstrain or a viral strain. In a related embodiment, the disease agent isa Gram-negative strain or a Gram positive strain.

The bacterium in various embodiments of the method is selected from thegroup consisting of: B. anthracis, B. cereus, C. botulinum, C.difficile, C. perfringens, C. spiroforme, and V. cholera, BI/NAP1/027and the J strain.

In certain embodiments, the binding regions bind to different diseaseagents, such that the binding protein is specific for a plurality ofdisease agents, e.g., a Clostridium toxin and an Escherichia toxin. Forexample, the binding protein includes a chimeric fusion protein specificto at least two different disease agents described herein. In certainembodiments of the method, the binding protein is a humanized antibodyderived from a non-human species for example a mouse, a rabbit, analpaca, a llama, or horse.

In a related embodiment, the method further includes observingneutralizing of the disease agent by the binding protein and/or survivalof the subject. In certain embodiments of the method, observing furtherincludes measuring an amount of the disease agent or a disease agentproduct in a sample from the subject. In various embodiments, the sampleis selected from: a cell, a fluid, and a tissue. For example, the fluidis at least one selected from: blood, serum, plasma, mucosal fluid,saliva, cerebrospinal fluid, semen, tears, and urine. In certainembodiments of the method, the cell or the tissue is at least oneselected from: fecal; vascular; epithelial; endothelial; dermal; dental;connective; muscular; neuronal; facial; cranial; soft tissue includingcartilage and collagen; brain; bone; bone marrow; joint tissue; andarticular joints. For example, the method includes collecting the fluid,the cell, or the tissue from a biopsy. In certain embodiments, themethod includes collecting the fluid, the cell, or the tissue from an exvivo sample or aliquot. Alternatively, the method includes collectingfrom fluid, cell, or tissue that is in vivo or in situ.

The method further includes in a related embodiment observing areduction or a remediation in at least one pathology symptom associatedwith the disease agent. In various embodiments, the method furtherincludes prior to contacting the subject with the binding protein,observing and/or detecting in the subject an indicium of the exposure tothe disease agent selected from: diarrhea, vomiting, breathingdifficulty, fever, inflammation, bleeding, pain, numbness, loss ofconsciousness, tissue necrosis, or organ failure. For example, thesubject is a transplant recipient or an immunosuppressed patient.

In a related embodiment, the method further includes contacting thesubject with the binding protein at a period of time such as seconds,minutes, or hours after observing the indicium. Alternatively, themethod further includes contacting the subject with the binding proteinseconds, minutes, hours, or days prior to an event that is associatedwith the risk for the exposure. For example, the method includescontacting the subject prior to or after the subject's entering apotentially hazardous or dangerous environment such as biohazardfacility, a combat zone, or a hazardous waste site.

The method in related embodiments includes contacting the subject withthe binding protein by injecting a solution including the bindingprotein into the subject. In various embodiments, injecting involves atleast one selected from: subcutaneous, intravenous, intramuscular,intraperitoneal, intradermal, intramedullary, transcutaneous, andintravitreal. In various embodiments of the method, contacting thesubject with binding protein includes at least one technique selectedfrom: topically, ocularly, nasally, bucally, orally, rectally,parenterally, intracisternally, intravaginally, or intraperitoneally. Ina related embodiment, contacting the subject involves using anapplicator, for example the applicator is a syringe, a needle, asprayer, a sponge, a gel, a strip, a tape, a bandage, a tray, a string,or a device used to apply a solution to a cell or a tissue. In variousembodiments, the pharmaceutical compositions (and/or additionaltherapeutic agents) are administered into the GI tract via, for example,oral delivery, nasogastral tube, intestinal intubation (e.g. an enteraltube or feeding tube such as, for example, a jejunal tube orgastro-jejunal tube, etc.), endoscopy, colonoscopy, or enema.

Some embodiments, provide a method of treating or preventing anantibiotic-induced adverse effect in the GI tract, comprisingadministering an effective amount of one or more pharmaceuticalcompositions described herein to a patient in need thereof (e.g. onereceiving or likely to receive one or more antibiotic treatments, e.g.one or more of fluoroquinolones, cephalosporins, clindamycin andpenicillins). In some embodiments, the antibiotic-induced adverse effectis associated with use of one or more of fluoroquinolones,cephalosporins, clindamycin and penicillins. Some embodiments, provide amethod of maintaining a normal intestinal micrbiota, comprisingadministering an effective amount of one or more pharmaceuticalcompositions described herein to a patient in need thereof (e.g. onereceiving or likely to receive one or more antibiotic treatments, e.g.one or more of fluoroquinolones, cephalosporins, clindamycin andpenicillins). Some embodiments, provide a method of preventing anovergrowth of one or more pathogenic microorganisms in the GI tract of apatient, comprising administering an effective amount of one or morepharmaceutical compositions described herein to a patient in needthereof (e.g. one receiving or likely to receive one or more antibiotictreatments, e.g. one or more of fluoroquinolones, cephalosporins,clindamycin and penicillins). Some embodiments, provide a method oftreating or preventing a C. difficile infection (CDI) or C.difficile-associated disease, comprising administering an effectiveamount of one or more pharmaceutical compositions described herein to apatient in need thereof (e.g. one receiving or likely to receive one ormore antibiotic treatments, e.g. one or more of fluoroquinolones,cephalosporins, clindamycin and penicillins). In various embodiments,the CDI and/or C. difficile associated disease is treated in the contextof initial onset or relapse. In various embodiments, the presentcompositions neutralize a C. difficile toxin (e.g toxin A and/or B).

In various embodiments, the CDI and/or C. difficile associated diseaseis one or more of antibiotic-induced adverse effect and/or CDI or C.difficile-associated disease is one or more of: antibiotic-associateddiarrhea, C. difficile diarrhea (CDD), C. difficile intestinalinflammatory disease, colitis, pseudomembranous colitis, fever,abdominal pain, dehydration and disturbances in electrolytes, megacolon,peritonitis, and perforation and/or rupture of the colon. In someembodiments, the present compositions and methods prevent or reducedilatation, mesocolonic edema, multifocal hemorrhages, and thickening ofthe intestinal wall. In various embodiments, including treatment and/orprevention of, for example an antibiotic-induced adverse effect in theGI tract, a ceftriaxone-associated adverse effect, an overgrowth of oneor more pathogenic microorganisms in the GI tract of a patient, a C.difficile infection (CDI) or C. difficile-associated disease andmaintenance of a normal intestinal micrbiota, the pharmaceuticalcomposition is any one of those described herein such as, by way ofnon-limiting example, pharmaceutical compositions comprising one or morerecombinant binding protein comprising at least one disease agentbinding domain amino acid sequence selected from SEQ ID NO: 174, SEQ IDNO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 171,SEQ ID NO: 172, and SEQ ID NO: 173, or variants thereof. In someembodiments, the recombinant binding protein comprising at least onedisease agent binding domain comprises a recombinant camelidheavy-chain-only antibody (VHH).

In various embodiments, the present methods treat or prevent anosocomial infection and/or a secondary emergent infection. In variousembodiments, the patient is undergoing treatment or has recentlyundergone treatment with one or more primary antibiotic (e.g. one ormore of fluoroquinolones, cephalosporins, clindamycin and penicillins).In various embodiments, the patient is undergoing treatment or hasrecently undergone treatment with one or more initial and/or adjunctivetherapy. In various embodiments, the an initial and/or adjunctivetherapy, selected from one or more of metronidazole, vancomycin,fidaxomicin, rifaximin, fecal bacteriotherapy, probiotic therapy, andantibody therapy, is administered to the patient.

In some embodiments, the present methods pertain to co-treatment (e.g.simultaneously or sequentially) with the pharmaceutical compositions ofthe present invention and/or any initial and/or adjunctive therapy. Inanother embodiment, the present methods pertain to treatment with aco-formulation of the pharmaceutical compositions of the presentinvention and any initial and/or adjunctive therapy. In otherembodiments, the present methods pertain to treating a C. difficileinfection (CDI) and/or a C. difficile-associated disease in a patientundergoing treatment with any additional agent described herein and/orany initial and/or adjunctive therapy described herein by administeringa pharmaceutical composition of the present invention to the patient.

In various embodiments, a CDI and/or C. difficile associated disease isprevented by administration of the present pharmaceutical compositionsto a patient that is at risk for CDI and/or C. difficile associateddisease (e.g. is undergoing or will undergoing antibiotic treatment,including IV antibiotic treatment and/or has previously been afflictedwith CDI and/or C. difficile associated disease). In variousembodiments, the CDI and/or C. difficile associated disease is treatedor prevented in the context of initial onset or relapse/recurrence (e.g.due to continued or restarted antibiotic therapy). For example, in apatient that has previously suffered from CDI, the presentpharmaceutical compositions (and/or additional agents) may beadministered upon the first symptoms of recurrence. By way ofnon-limiting example, symptoms of recurrence include, in a mild case,about 5 to about 10 watery bowel movements per day, no significantfever, and only mild abdominal cramps while blood tests may show a mildrise in the white blood cell count up to about 15,000 (normal levels areup to about 10,000), and, in a severe case, more than about 10 waterystools per day, nausea, vomiting, high fever (e.g. about 102-104° F.),rectal bleeding, severe abdominal pain (e.g. with tenderness), abdominaldistention, and a high white blood count (e.g. of about 15,000 to about40,000).

Regardless of initial onset or relapse/recurrence, CDI and/or C.difficile associated disease may be diagnosed via any of the symptomsdescribed herein (e.g. watery diarrhea about 3 or more times a day forabout 2 days or more, mild to bad cramping and pain in the belly, fever,blood or pus in the stool, nausea, dehydration, loss of appetite, lossof weight, etc.). Regardless of initial onset or relapse/recurrence, CDIand/or C. difficile associated disease may also be diagnosed via enzymeimmunoassays e.g. to detect the C. difficile toxin A or B antigen and/orglutamine dehydrogenase (GDH), which is produced by C. difficileorganisms, polymerase chain reaction (e.g. to detect the C. difficiletoxin A or B gene or a portion thereof (e.g. tcdA or tcdB), includingthe ILLUMIGENE LAMP assay), a cell cytotoxicity assay. For example, anyone of the following tests may be used may be used: Meridian ImmunoCardToxins A/B; Wampole Toxin A/B Quik Chek; Wampole C. difficile Quik ChekComplete; Remel Xpect Clostridium difficile Toxin A/B; Meridian PremierToxins A/B; Wampole C. difficile Tox A/B II; Remel Prospect Toxin A/BEIA; Biomerieux Vidas C. difficile Toxin A&B; BD Geneohm C. diff;Prodesse Progastro CD; and Cepheld Xpert C. difficile In variousembodiments, the clinical sample is a patient stool sample. Also aflexible sigmoidoscopy “scope” test and/or an abdominal X-ray and/or acomputerized tomography (CT) scan, which provides images of the colon,may be used in assessing a patient (e.g. looking for characteristiccreamy white or yellow plaques adherent to the wall of the colon).Further, biopsies (e.g. of any region of the GI tract) may be used toassess a potential CDI and/or C. difficile associated disease patient.

Furthermore, the patients of the invention include, but are not limitedto, patients that are at a particular risk for CDI and/or C. difficileassociated disease, such as those which have been taking an antibioticduring the past 30 or so days and/or have an immune system that is weak(e.g. from a chronic illness) and/or are women and/or are elderly (e.g.over about 65 years old) and/or are elderly woman and/or undergotreatment with for heartburn or stomach acid disorders (e.g. with agentssuch as PREVACID, TAGAMET, PRILOSEC, or NEXIUM and related drugs) and/orhave recently been in the hospital, including in an intensive care unit,or live in a nursing home. Accordingly, in some embodiments, thepharmaceutical composition of the present invention may be used toprophylactically prevent CDI and/or C. difficile associated disease.

In some embodiments, the methods and uses of the present inventionrelate to a patient is undergoing treatment or has recently undergonetreatment with one or more primary antibiotic. A “primary antibiotic”refers to an antibiotic that is administered to a patient and which mayresult in CDI and/or C. difficile associated disease. These include theantibiotics that most often lead to CDI and/or C. difficile associateddisease, such as, for example, fluoroquinolones, cephalosporins,clindamycin and penicillins.

In some embodiments, the methods and uses of the present inventioninclude those in which an initial and/or adjunctive therapy isadministered to a patient. Initial and/or adjunctive therapy indicatestherapy that is used to treat CDI and/or C. difficile associated diseaseupon detection of such disease. In some embodiments, the initial and/oradjunctive therapy is one or more of metronidazole, vancomycin,fidaxomicin, rifaximin, fecal bacteriotherapy, probiotic therapy, acharcoal-based therapy, and antibody therapy, as described herein. Invarious embodiments, the methods and uses of the present inventioninclude use of the inventive pharmaceutical composition as an adjuvantto any of these initial and/or adjunctive therapies (includingco-administration or sequential administration). In various embodiments,the methods and uses of the present invention include use of theinventive pharmaceutical composition in a patient undergoing initialand/or adjunctive therapies.

In a related embodiment of the method, contacting the subject with thebinding protein includes administering to the subject a source ofexpression of the binding protein. In various embodiments of the method,the source of expression of the binding protein is a nucleotide sequenceencoding the binding protein, such that the source of the expressionincludes at least one selected from the group consisting of: a nakednucleic acid vector, bacterial vector, and a viral vector. For example,the bacterial vector is derived from at least one selected from thegroup consisting of: E. coli, Bacillus spp, Clostridium spp,Lactobacillus spp, and Lactococcus spp.

In a related embodiment of the method, contacting further includesadministering the vector, for example the naked nucleic acid vector, thebacterial vector, or the viral vector.

In a related embodiment, the nucleotide acid sequence further includesan operably linked signal for promoting expression of the bindingprotein. For example, the signal includes a mammalian promoter or anon-viral promoter. In a related embodiment, the method involvesengineering the binding protein or the source of expression of thebinding protein (e.g., viral vector or bacterial vector) using adimerizer sequence for example having an amino acid sequence includingSEQ ID NO: 94 or a portion or homolog or variant thereof. For example,the dimerizer sequences forms a covalent bond or disulfide linkagebetween at least two amino acid sequences to form a homodimer, aheterodimer, or a multimer. The method in various embodiments includes,prior to contacting, engineering the binding protein using an agent thatmultimerizes at least one binding region or a multimer, e.g., aheterodimer, a heterotrimer, and a heterotetramer, to form the bindingprotein.

In a related embodiment of the method, the viral vector is derived fromat least one selected from: an adenovirus, an adeno-associated virus, aherpesvirus, and a lentivirus. The method in various embodiments furtherincludes contacting the subject with a gene delivery vehicle selectedfrom at least one of: a liposome, a lipid/polycation (LPD), a peptide, ananoparticle, a gold particle, and a polymer. For example, the genedelivery vehicle specifically targets a cell or tissue in the body bycontacting or binding a receptor located on the cell or tissue.

An aspect of the invention provides a pharmaceutical composition fortreating a subject at risk for exposure to or exposed to a diseaseagent, the pharmaceutical composition including: at least onerecombinant heteromultimeric neutralizing binding protein including twoor more binding regions, such that the binding regions are notidentical, and each binding region specifically binds a non-overlappingportion of the disease agent, such that the binding protein neutralizesthe disease agent, thereby treating the subject for exposure to thedisease agent.

In a related embodiment, the composition is compounded with apharmaceutically acceptable buffer or diluent. For example, thecomposition is compounded for parenteral administration such asintravenous, mucosal administration, topical administration, or oraladministration.

In various embodiments, the subject is at least one selected from: ahuman, a dog, a cat, a goat, a cow, a pig, and a horse. For example, thehuman subject is a: sick child or adult, healthcare profession (e.g.,doctor and nurse), aid worker, member of the military, or animmunosuppressed patient such as a transplant recipient. In someembodiments, the subject is a hospital patient at risk for a HospitalAcquired Infections (or Healthcare Acquired Infections, HAI). In someembodiments, the subject is one receiving or likely to receive one ormore antibiotic treatments, e.g. one or more of fluoroquinolones,cephalosporins, clindamycin and penicillins. In certain embodiments, thepharmaceutical composition is formulated to protect the subject againstthe exposure, for example that exposure includes a picogram amount,nanogram amount, microgram amount, or gram amount of the disease agentor a plurality of disease agents.

The binding protein or binding regions in various embodiments of thecomposition is selected from the group of: a single-chain antibody(scFv); a recombinant camelid heavy-chain-only antibody (VHH); a sharkheavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Sac7d derivative (affitins, e.g.NANOFITINS), a Fv; a Fab; a Fab′; and a F(ab′)₂. In various embodiments,the binding regions are of a different type, for example at least onebinding region is a VHH and at least other binding region is a scFv, anFab or any of the types described herein.

The composition in various embodiments further includes at least oneagent selected from the group of: an antitoxin, an anti-inflammatory, ananti-tumor, an antiviral, an antibacterial, an anti-mycobacterial, ananti-fungal, an anti-proliferative, an anti-apoptotic, an anti-allergy,and an anti-immune suppressant.

In an embodiment, the composition further includes a labeled detectablemarker selected from the group consisting of: detectable, fluorescent,colorimetric, enzymatic, radioactive, and the like. For example, themarker is detectable in a sample taken from the subject, the sampleexemplified by a cell, a fluid or a tissue. In a related embodiment, themarker includes a peptide, a protein, a carbohydrate, and a polymer.

In an embodiment of the composition, the binding protein includes alinker that separates the binding regions. The linker in a relatedembodiment separates the binding regions and/or subunits of themultimeric protein. In certain embodiments, the binding protein includesa linker that covalently joins each binding region of the heterodimericor the multimeric protein. In various embodiments, the linker includesat least one selected from the group of: a peptide, a protein, a sugar,or a nucleic acid. In a related embodiment, the linker includes aminoacid sequence GGGGS (SEQ ID NO: 54) or a portion or variant thereof. Ina related embodiment, the linker includes amino acid sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 55) or a portion or variant thereof ormultiples thereof. The linker in various embodiments stabilizes thebinding protein and does not prevent the respective binding of thebinding regions to the disease agent or to a plurality of diseaseagents.

In various embodiments of the pharmaceutical composition, the bindingprotein and/or binding regions include at least one tag that is attachedor genetically fused to the binding protein and/or binding regions. Thetag for example is a peptide, sugar, or DNA molecule that does notinhibit or prevent binding of the binding protein and/or binding regionsto the disease agent. In various embodiments, the tag is at least about:three to five amino acids long, five to eight amino acids long, eight totwelve amino acids long, twelve to fifteen amino acids long, or fifteento twenty amino acids long. For example, the tag includes SEQ ID NO: 15,or a variant thereof.

In various embodiments, the disease agent for which the binding proteinis specific is at least one selected from: a virus, a cancer cell, afungus, a bacterium, a parasite and a product thereof such as apathogenic molecule, a protein, a lipopolysaccharide, or a toxin. Inrelated embodiments of the composition, the toxin includes a protein, alipid, a lipopolysaccharide, and a small molecule toxin such as anaflatoxin or a dinoflagellate toxin. For example, the toxin is aBotulinum neurotoxin comprising a serotype selected from: A, B, C, D, E,F, and G. In various embodiments of the composition, the toxin is atleast one selected from: staphylococcal α-hemolysin, staphylococcalleukocidin, aerolysin cytotoxic enterotoxin, a cholera toxin, Bacilluscereus hemolysis II toxin, a Helicobacter pylori vacuolating toxin, aBacillus anthracis toxin, a cholera toxin, a Escherichia coli serotypeO157:H7 toxin, a Escherichia coli serotype 0104:H7 toxin, alipopolysaccharide endotoxin, a Shiga toxin, a pertussis toxin, aClostridium perfringens iota toxin, a Clostridium spiroforme toxin, aClostridium difficile toxin A, a Clostridium difficile toxin B, aClostridium septicum a toxin, and a Clostridium botulinum C2 toxin. Incertain embodiments, the disease agent includes a plurality ofnon-identical disease agents such that the binding regions of thebinding protein bind to and neutralize the plurality of disease agents.

In various embodiments of the composition, the bacterium for which thebinding protein is specific is selected from: B. anthracis, B. cereus,C. botulinum, C. difficile, C. perfringens, V. cholerae, and C.spiroforme. In a related embodiment, the bacterium is a virulentbacterium or apathogenic bacterium.

The composition in various embodiments is compounded or formulated for aroute of delivery selected from the group of: topical, ocular, nasal,bucal, oral, rectal, parenteral, intracisternal, invaginal, andintraperitoneal.

In various embodiments of the composition, the binding protein isspecific for a toxin which is a C. botulinum toxin, and the bindingregions of the binding protein includes a recombinant camelidheavy-chain-only antibody, and the composition includes an amino acidsequence selected from the group:

(VHH H7, SEQ ID NO: 56) LVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQ GTQVTVSSAHHSEDP;(VHH B5, SEQ ID NO: 57) LVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHY WGKGTLVTVSSEPKTPKPQ;and (H7/B5 heterodimer, SEQ ID NO: 58)QVQLVESGGGLVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDPTSAIAGGGGSGGGGSGGGGSLQGQLQLVESGGGLVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ, or variants thereof.

In a related embodiment of the composition, the binding protein isspecific for a toxin which is a C. difficile toxin A, and the bindingregion of the binding protein includes a recombinant camelidheavy-chain-only antibody having an amino acid sequence selected fromthe group of:

(AH3, SEQ ID NO: 59) QVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQP; (AA6, SEQ ID NO: 60)QLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQP; (A3H, SEQ ID NO: 61)QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSVDGSTYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQSPIPIHYSRTYSGPYGMDYWGKGTLVTVSSAHHSEDP; (AC1, SEQ ID NO: 62)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISFVDGSTYYADSVKGRFAISRGNAKNTVYLQMNSLKPEDTAVYYCAADQSSIPMHYSSTYSGPSGMDYWGKGTLVTVSSEPKTPKPQP; (A11G, SEQ ID NO: 63)QLQLVETGGGLVQAGGSLRLSCAASGRTLSNYPMGWFRQAPGKEREFVAAIRRIADGTYYADSVKGRFTISRDNAWNTLYLQMNGLKPEDTAVYFCATGPGAFPGMVVTNPSAYPYWGQGTQVTVSSEPKTPKPQP; (AE1, SEQ ID NO: 64)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSSDGSTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAADQAAIPMHYSASYSGPRGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 87)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSAIAGGGGSGGGGSGGGGSLQAMAAASQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRG QGIQVTVSSEPKTPKPQPARR;and, (SEQ ID NO: 95) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSLQAMAAAQLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC, or variants thereof.

In certain embodiments of the composition, the binding protein isspecific for a toxin which is a C. difficile toxin B, and the bindingregion of the binding protein includes a recombinant camelidheavy-chain-only antibody having an amino acid sequence selected fromthe group consisting of:

(2D, SEQ ID NO: 65) QVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGIGWFRQAPGKERQEVSYISASAKTKLYSDSVKGRFTISRDNAKNAVYLEMNSLKREDTAVYYCARRRFDASASNRWLAADYDYWGQGTQVTVSSEPKTPKPQ; (2Ds, SEQ ID NO: 66)QVQLVESGGGLVQAGGSLRLSCVSSERNPGINAMGWYRQAPGSQRELVAIWQTGGSLNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDQYWGQGTQVTVSSEPKTPKPQ; (5D, SEQ ID NO: 67)QVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQ; (E3, SEQ ID NO: 68)QVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ; (7F, SEQ ID NO: 69)QVQLVESGGGLVEAGGSLRLSCVVTGSSFSTSTMAWYRQPPGKQREWVASFTSGGAIKYTDSVKGRFTMSRDNAKKMTYLQMENLKPEDTAVYYCALHNAVSGSSWGRGTQVTVSSEPKTPKPQ; (5E, SEQ ID NO: 70)VQLVESGGGLVQAGGSLRLSCAASGLMFGAMTMGWYRQAPGKEREMVAYITAGGTESYSESVKGRFTISRINANNMVYLQMTNLKVEDTAVYYCNAHNFWRTSRNWGQGTQVTVSSEPKTPKP; (B12, SEQ ID NO: 71)VQLVESGGGLVQAGDSLTLSCAASESTFNTFSMAWFRQAPGKEREYVAAFSRSGGTTNYADSVKGRATISTDNAKNTVYLHMNSLKPEDTAVYFCAADRPAGRAYFQSRSYNYWGQGTQVTVSSAHHSEDP; (A11, SEQ ID NO: 72)VQLVESGGGSVQIGGSLRLSCVASGFTFSKNIMSWARQAPGKGLEWVSTISIGGAATSYADSVKGRFTISRDNANDTLYLQMNNLKPEDTAVYYCSRGPRTYINTASRGQGTQVTVSSEPKTPKP; (AB8, SEQ ID NO: 73)VQLVESGGGLVQAGGSLRLSCVGSGRNPGINAMGWYRQAPGSQRELVAVWQTGGSTNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDEYWGQGTQVTVSSAHHSEDP; (C6, SEQ ID NO: 74)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPKTPKP; (C12, SEQ ID NO: 75)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPKTP; (A1, SEQ ID NO: 76)VQLVESGGGLVQAGGSLRLSCAAPGLTFTSYRMGWFRQAPGKEREYVAAITGAGATNYADSAKGRFTISKNNTASTVHLQMNSLKPEDTAVYYCAASNRAGGYWRASQYDYWGQGTQVTVSSAHHSEDP; SEQ ID NO: 87; and SEQ ID NO: 95,or variants thereof.

In some embodiments, the present compositions, methods and kits pertainto one or more multimers, e.g. dimeric, trimer or tetrameric constructsof binding proteins specific for a toxin which is a C. difficile toxin Aand/or a C. difficile toxin B, and the binding region of the bindingprotein optionally including a recombinant camelid heavy-chain. In someembodiments, the multimers are linked via any of the linkers disclosedherein (e.g. GGGGS (SEQ ID NO: 54), or GGGGSGGGGSGGGGS (SEQ ID NO: 55),or a portion or variant thereof). In some embodiments, the presentcompositions, methods and kits pertain to one or more multimers, e.g.dimeric, trimer or tetrameric constructs of binding proteins specificfor a toxin which is a C. difficile toxin A and/or a C. difficile toxinB, and the binding region of the binding protein optionally including arecombinant camelid heavy-chain that are substantiallyprotease-resistant, e.g. are stable in the GI tract. In someembodiments, the constructs have a reduced amount or are substantiallyfree of alanine residues that are susceptible to proteolysis. In someembodiments, the multimers are linked via any of the linkers disclosedherein (e.g. GGGGS (SEQ ID NO: 54), or GGGGSGGGGSGGGGS (SEQ ID NO: 55),or a portion or variant thereof).

In some embodiments, the binding proteins specific for a C. difficiletoxin A include one or more of AH3, e.g. SEQ ID NO: 59, AA6, e.g. SEQ IDNO: 60, A3H, e.g. SEQ ID NO: 61, AC1, e.g. SEQ ID NO: 62, A11G, e.g. SEQID NO: 63, and AE1, e.g. SEQ ID NO: 64. In some embodiments, the bindingproteins specific for a C. difficile toxin B include one or more of 2D,e.g. SEQ ID NO: 65, 2Ds, e.g. SEQ ID NO: 66, 5D, e.g. SEQ ID NO: 67, E3,e.g. SEQ ID NO: 68, 7F, e.g. SEQ ID NO: 69, 5E, e.g. SEQ ID NO: 70, B12,e.g. SEQ ID NO: 71, A11, e.g. SEQ ID NO: 72, AB8, e.g. SEQ ID NO: 73,C6, e.g. SEQ ID NO: 74, C12, e.g. SEQ ID NO: 75, and A1, e.g. SEQ ID NO:76, or variants thereof.

In some embodiments, such multimers may include dimers. For example, thedimers may be homo- or hetero-dimers. Illustrative dimers are twobinding proteins specific for a C. difficile toxin A, including by wayof non-limitation, AH3, e.g. SEQ ID NO: 59/AH3, e.g. SEQ ID NO: 59; AA6,e.g. SEQ ID NO: 60/AH3, e.g. SEQ ID NO: 59; A3H, e.g. SEQ ID NO: 61/AH3,e.g. SEQ ID NO: 59; AC1, e.g. SEQ ID NO: 62/AH3, e.g. SEQ ID NO: 59;A11G, e.g. SEQ ID NO: 63/AH3, e.g. SEQ ID NO: 59; AE1, e.g. SEQ ID NO:64/AH3, e.g. SEQ ID NO: 59; AH3, e.g. SEQ ID NO: 59/AA6, e.g. SEQ ID NO:60; AA6, e.g. SEQ ID NO: 60, /AA6, e.g. SEQ ID NO: 60; A3H, e.g. SEQ IDNO: 61/AA6, e.g. SEQ ID NO: 60; AC1, e.g. SEQ ID NO: 62/AA6, e.g. SEQ IDNO: 60; A11G, e.g. SEQ ID NO: 63/AA6, e.g. SEQ ID NO: 60; AE1, e.g. SEQID NO: 64/AA6, e.g. SEQ ID NO: 60; AH3, e.g. SEQ ID NO: 59/A3H, e.g. SEQID NO: 61; AA6, e.g. SEQ ID NO: 60/A3H, e.g. SEQ ID NO: 61; A3H, e.g.SEQ ID NO: 61/A3H, e.g. SEQ ID NO: 61; AC1, e.g. SEQ ID NO: 62/A3H, e.g.SEQ ID NO: 61; A11G, e.g. SEQ ID NO: 63/A3H, e.g. SEQ ID NO: 61; AE1,e.g. SEQ ID NO: 64/A3H, e.g. SEQ ID NO: 61; AH3, e.g. SEQ ID NO: 59/AC1,e.g. SEQ ID NO: 62; AA6, e.g. SEQ ID NO: 60/AC1, e.g. SEQ ID NO: 62;A3H, e.g. SEQ ID NO: 61/AC1, e.g. SEQ ID NO: 62; AC1, e.g. SEQ ID NO:62/AC1, e.g. SEQ ID NO: 62; A11G, e.g. SEQ ID NO: 63/AC1, e.g. SEQ IDNO: 62; AE1, e.g. SEQ ID NO: 64/AC1, e.g. SEQ ID NO: 62; AH3, e.g. SEQID NO: 59/A11G, e.g. SEQ ID NO: 63; AA6, e.g. SEQ ID NO: 60/A11G, e.g.SEQ ID NO: 63; A3H, e.g. SEQ ID NO: 61/A11G, e.g. SEQ ID NO: 63; AC1,e.g. SEQ ID NO: 62/A11G, e.g. SEQ ID NO: 63; A11G, e.g. SEQ ID NO:63/A11G, e.g. SEQ ID NO: 63; AE1, e.g. SEQ ID NO: 64/A11G, e.g. SEQ IDNO: 63; AH3, e.g. SEQ ID NO: 59/AE1, e.g. SEQ ID NO: 64; AA6, e.g. SEQID NO: 60/AE1, e.g. SEQ ID NO: 64; A3H, e.g. SEQ ID NO: 61/AE1, e.g. SEQID NO: 64; AC1, e.g. SEQ ID NO: 62/AE1, e.g. SEQ ID NO: 64; A1 1G, e.g.SEQ ID NO: 63/AE1, e.g. SEQ ID NO: 64; and AE1, e.g. SEQ ID NO: 64/AE1,e.g. SEQ ID NO: 64, or variants thereof.

In some embodiments, such multimers may include dimers. For example, thedimers may be homo- or hetero-dimers. Illustrative dimers are twobinding proteins specific for a C. difficile toxin B, including by wayof non-limitation, 2D, e.g. SEQ ID NO: 65/2D, e.g. SEQ ID NO: 65, 2Ds,e.g. SEQ ID NO: 66/2D, e.g. SEQ ID NO: 65, 5D, e.g. SEQ ID NO: 67/2D,e.g. SEQ ID NO: 65, E3, e.g. SEQ ID NO: 68/2D, e.g. SEQ ID NO: 65, 7F,e.g. SEQ ID NO: 69/2D, e.g. SEQ ID NO: 65, 5E, e.g. SEQ ID NO: 70/2D,e.g. SEQ ID NO: 65, B12, e.g. SEQ ID NO: 71/2D, e.g. SEQ ID NO: 65, A11,e.g. SEQ ID NO: 72/2D, e.g. SEQ ID NO: 65, AB8, e.g. SEQ ID NO: 73/2D,e.g. SEQ ID NO: 65, C6, e.g. SEQ ID NO: 74/2D, e.g. SEQ ID NO: 65, C12,e.g. SEQ ID NO: 75/2D, e.g. SEQ ID NO: 65, A1, e.g. SEQ ID NO: 76/2D,e.g. SEQ ID NO: 65, 2D, e.g. SEQ ID NO: 65/2Ds, e.g. SEQ ID NO: 66, 2Ds,e.g. SEQ ID NO: 66/2Ds, e.g. SEQ ID NO: 66, 5D, e.g. SEQ ID NO: 67/2Ds,e.g. SEQ ID NO: 66, E3, e.g. SEQ ID NO: 68/2Ds, e.g. SEQ ID NO: 66, 7F,e.g. SEQ ID NO: 69/2Ds, e.g. SEQ ID NO: 66, 5E, e.g. SEQ ID NO: 70/2Ds,e.g. SEQ ID NO: 66, B12, e.g. SEQ ID NO: 71/2Ds, e.g. SEQ ID NO: 66,A11, e.g. SEQ ID NO: 72/2Ds, e.g. SEQ ID NO: 66, AB8, e.g. SEQ ID NO:73/2Ds, e.g. SEQ ID NO: 66, C6, e.g. SEQ ID NO: 74/2Ds, e.g. SEQ ID NO:66, C12, e.g. SEQ ID NO: 75/2Ds, e.g. SEQ ID NO: 66, A1, e.g. SEQ ID NO:76/2Ds, e.g. SEQ ID NO: 66, 2D, e.g. SEQ ID NO: 65/5D, e.g. SEQ ID NO:67, 2Ds, e.g. SEQ ID NO: 66/5D, e.g. SEQ ID NO: 67, 5D, e.g. SEQ ID NO:67/5D, e.g. SEQ ID NO: 67, E3, e.g. SEQ ID NO: 68/5D, e.g. SEQ ID NO:67, 7F, e.g. SEQ ID NO: 69/5D, e.g. SEQ ID NO: 67, 5E, e.g. SEQ ID NO:70/5D, e.g. SEQ ID NO: 67, B12, e.g. SEQ ID NO: 71/5D, e.g. SEQ ID NO:67, A11, e.g. SEQ ID NO: 72/5D, e.g. SEQ ID NO: 67, AB8, e.g. SEQ ID NO:73/5D, e.g. SEQ ID NO: 67, C6, e.g. SEQ ID NO: 74/5D, e.g. SEQ ID NO:67, C12, e.g. SEQ ID NO: 75/5D, e.g. SEQ ID NO: 67, A1, e.g. SEQ ID NO:76/5D, e.g. SEQ ID NO: 67, 2D, e.g. SEQ ID NO: 65/E3, e.g. SEQ ID NO:68, 2Ds, e.g. SEQ ID NO: 66/E3, e.g. SEQ ID NO: 68, 5D, e.g. SEQ ID NO:67/E3, e.g. SEQ ID NO: 68, E3, e.g. SEQ ID NO: 68/E3, e.g. SEQ ID NO:68, 7F, e.g. SEQ ID NO: 69/E3, e.g. SEQ ID NO: 68, 5E, e.g. SEQ ID NO:70/E3, e.g. SEQ ID NO: 68, B12, e.g. SEQ ID NO: 71/E3, e.g. SEQ ID NO:68, A11, e.g. SEQ ID NO: 72/E3, e.g. SEQ ID NO: 68, AB8, e.g. SEQ ID NO:73/E3, e.g. SEQ ID NO: 68, C6, e.g. SEQ ID NO: 74/E3, e.g. SEQ ID NO:68, C12, e.g. SEQ ID NO: 75/E3, e.g. SEQ ID NO: 68, A1, e.g. SEQ ID NO:76/E3, e.g. SEQ ID NO: 68, 2D, e.g. SEQ ID NO: 65/7F, e.g. SEQ ID NO:69, 2Ds, e.g. SEQ ID NO: 66/7F, e.g. SEQ ID NO: 69, 5D, e.g. SEQ ID NO:67/7F, e.g. SEQ ID NO: 69, E3, e.g. SEQ ID NO: 68/7F, e.g. SEQ ID NO:69, 7F, e.g. SEQ ID NO: 69/7F, e.g. SEQ ID NO: 69, 5E, e.g. SEQ ID NO:70/7F, e.g. SEQ ID NO: 69, B12, e.g. SEQ ID NO: 71/7F, e.g. SEQ ID NO:69, A11, e.g. SEQ ID NO: 72/7F, e.g. SEQ ID NO: 69, AB8, e.g. SEQ ID NO:73/7F, e.g. SEQ ID NO: 69, C6, e.g. SEQ ID NO: 74/7F, e.g. SEQ ID NO:69, C12, e.g. SEQ ID NO: 75/7F, e.g. SEQ ID NO: 69, A1, e.g. SEQ ID NO:76/7F, e.g. SEQ ID NO: 69, 2D, e.g. SEQ ID NO: 65/5E, e.g. SEQ ID NO:70, 2Ds, e.g. SEQ ID NO: 66/5E, e.g. SEQ ID NO: 70, 5D, e.g. SEQ ID NO:67/5E, e.g. SEQ ID NO: 70, E3, e.g. SEQ ID NO: 68/5E, e.g. SEQ ID NO:70, 7F, e.g. SEQ ID NO: 69/5E, e.g. SEQ ID NO: 70, 5E, e.g. SEQ ID NO:70/5E, e.g. SEQ ID NO: 70, B12, e.g. SEQ ID NO: 71/5E, e.g. SEQ ID NO:70, A11, e.g. SEQ ID NO: 72/5E, e.g. SEQ ID NO: 70, AB8, e.g. SEQ ID NO:73/5E, e.g. SEQ ID NO: 70, C6, e.g. SEQ ID NO: 74/5E, e.g. SEQ ID NO:70, C12, e.g. SEQ ID NO: 75/5E, e.g. SEQ ID NO: 70, A1, e.g. SEQ ID NO:76/5E, e.g. SEQ ID NO: 70, 2D, e.g. SEQ ID NO: 65/B12, e.g. SEQ ID NO:71, 2Ds, e.g. SEQ ID NO: 66/B12, e.g. SEQ ID NO: 71, 5D, e.g. SEQ ID NO:67/B12, e.g. SEQ ID NO: 71, E3, e.g. SEQ ID NO: 68/B12, e.g. SEQ ID NO:71, 7F, e.g. SEQ ID NO: 69/B12, e.g. SEQ ID NO: 71, 5E, e.g. SEQ ID NO:70/B12, e.g. SEQ ID NO: 71, B12, e.g. SEQ ID NO: 71/B12, e.g. SEQ ID NO:71, A1 1, e.g. SEQ ID NO: 72/B12, e.g. SEQ ID NO: 71, AB8, e.g. SEQ IDNO: 73/B12, e.g. SEQ ID NO: 71, C6, e.g. SEQ ID NO: 74/B12, e.g. SEQ IDNO: 71, C12, e.g. SEQ ID NO: 75/B12, e.g. SEQ ID NO: 71, A1, e.g. SEQ IDNO: 76/B12, e.g. SEQ ID NO: 71, 2D, e.g. SEQ ID NO: 65/A11, e.g. SEQ IDNO: 72, 2Ds, e.g. SEQ ID NO: 66/A11, e.g. SEQ ID NO: 72, 5D, e.g. SEQ IDNO: 67/A11, e.g. SEQ ID NO: 72, E3, e.g. SEQ ID NO: 68/A11, e.g. SEQ IDNO: 72, 7F, e.g. SEQ ID NO: 69/A11, e.g. SEQ ID NO: 72, 5E, e.g. SEQ IDNO: 70/A11, e.g. SEQ ID NO: 72, B12, e.g. SEQ ID NO: 71/A11, e.g. SEQ IDNO: 72, A11, e.g. SEQ ID NO: 72/A11, e.g. SEQ ID NO: 72, AB8, e.g. SEQID NO: 73/A11, e.g. SEQ ID NO: 72, C6, e.g. SEQ ID NO: 74/A11, e.g. SEQID NO: 72, C12, e.g. SEQ ID NO: 75/A11, e.g. SEQ ID NO: 72, A1, e.g. SEQID NO: 76/A11, e.g. SEQ ID NO: 72, 2D, e.g. SEQ ID NO: 65/AB8, e.g. SEQID NO: 73, 2Ds, e.g. SEQ ID NO: 66/AB8, e.g. SEQ ID NO: 73, 5D, e.g. SEQID NO: 67/AB8, e.g. SEQ ID NO: 73, E3, e.g. SEQ ID NO: 68/AB8, e.g. SEQID NO: 73, 7F, e.g. SEQ ID NO: 69/AB8, e.g. SEQ ID NO: 73, 5E, e.g. SEQID NO: 70/AB8, e.g. SEQ ID NO: 73, B12, e.g. SEQ ID NO: 71/AB8, e.g. SEQID NO: 73, A11, e.g. SEQ ID NO: 72/AB8, e.g. SEQ ID NO: 73, AB8, e.g.SEQ ID NO: 73/AB8, e.g. SEQ ID NO: 73, C6, e.g. SEQ ID NO: 74/AB8, e.g.SEQ ID NO: 73, C12, e.g. SEQ ID NO: 75/AB8, e.g. SEQ ID NO: 73, A1, e.g.SEQ ID NO: 76/AB8, e.g. SEQ ID NO: 73, 2D, e.g. SEQ ID NO: 65/C6, e.g.SEQ ID NO: 74, 2Ds, e.g. SEQ ID NO: 66/C6, e.g. SEQ ID NO: 74, 5D, e.g.SEQ ID NO: 67/C6, e.g. SEQ ID NO: 74, E3, e.g. SEQ ID NO: 68/C6, e.g.SEQ ID NO: 74, 7F, e.g. SEQ ID NO: 69/C6, e.g. SEQ ID NO: 74, 5E, e.g.SEQ ID NO: 70/C6, e.g. SEQ ID NO: 74, B12, e.g. SEQ ID NO: 71/C6, e.g.SEQ ID NO: 74, A11, e.g. SEQ ID NO: 72/C6, e.g. SEQ ID NO: 74, AB8, e.g.SEQ ID NO: 73/C6, e.g. SEQ ID NO: 74, C6, e.g. SEQ ID NO: 74/C6, e.g.SEQ ID NO: 74, C12, e.g. SEQ ID NO: 75/C6, e.g. SEQ ID NO: 74, A1, e.g.SEQ ID NO: 76/C6, e.g. SEQ ID NO: 74, 2D, e.g. SEQ ID NO: 65/C12, e.g.SEQ ID NO: 75, 2Ds, e.g. SEQ ID NO: 66/C12, e.g. SEQ ID NO: 75, 5D, e.g.SEQ ID NO: 67/C12, e.g. SEQ ID NO: 75, E3, e.g. SEQ ID NO: 68/C12, e.g.SEQ ID NO: 75, 7F, e.g. SEQ ID NO: 69/C12, e.g. SEQ ID NO: 75, 5E, e.g.SEQ ID NO: 70/C12, e.g. SEQ ID NO: 75, B12, e.g. SEQ ID NO: 71/C12, e.g.SEQ ID NO: 75, A11, e.g. SEQ ID NO: 72/C12, e.g. SEQ ID NO: 75, AB8,e.g. SEQ ID NO: 73/C12, e.g. SEQ ID NO: 75, C6, e.g. SEQ ID NO: 74/C12,e.g. SEQ ID NO: 75, C12, e.g. SEQ ID NO: 75/C12, e.g. SEQ ID NO: 75, A1,e.g. SEQ ID NO: 76/C12, e.g. SEQ ID NO: 75, 2D, e.g. SEQ ID NO: 65/A1,e.g. SEQ ID NO: 76, 2Ds, e.g. SEQ ID NO: 66/A1, e.g. SEQ ID NO: 76, 5D,e.g. SEQ ID NO: 67/A1, e.g. SEQ ID NO: 76, E3, e.g. SEQ ID NO: 68/A1,e.g. SEQ ID NO: 76, 7F, e.g. SEQ ID NO: 69/A1, e.g. SEQ ID NO: 76, 5E,e.g. SEQ ID NO: 70/A1, e.g. SEQ ID NO: 76, B12, e.g. SEQ ID NO: 71/A1,e.g. SEQ ID NO: 76, A11, e.g. SEQ ID NO: 72/A1, e.g. SEQ ID NO: 76, AB8,e.g. SEQ ID NO: 73/A1, e.g. SEQ ID NO: 76, C6, e.g. SEQ ID NO: 74/A1,e.g. SEQ ID NO: 76, C12, e.g. SEQ ID NO: 75/A1, e.g. SEQ ID NO: 76, andA1, e.g. SEQ ID NO: 76/A1, e.g. SEQ ID NO: 76, or variants thereof.

In some embodiments, such multimers may include dimers that are onebinding protein specific for a C. difficile toxin A and one bindingprotein specific for a C. difficile toxin B. For example, the dimers mayone monomer binding protein selected from AH3, e.g. SEQ ID NO: 59, AA6,e.g. SEQ ID NO: 60, A3H, e.g. SEQ ID NO: 61, AC1, e.g. SEQ ID NO: 62,A11G, e.g. SEQ ID NO: 63, and AE1, e.g. SEQ ID NO: 64 and monomerbinding protein selected from 2D, e.g. SEQ ID NO: 65, 2Ds, e.g. SEQ IDNO: 66, 5D, e.g. SEQ ID NO: 67, E3, e.g. SEQ ID NO: 68, 7F, e.g. SEQ IDNO: 69, 5E, e.g. SEQ ID NO: 70, B12, e.g. SEQ ID NO: 71, A11, e.g. SEQID NO: 72, AB8, e.g. SEQ ID NO: 73, C6, e.g. SEQ ID NO: 74, C12, e.g.SEQ ID NO: 75, and A1, e.g. SEQ ID NO: 76, or variants thereof.

In some embodiments, such multimers may include tetramers having atleast one monomer selected from one binding protein specific for a C.difficile toxin A and one binding protein specific for a C. difficiletoxin B. Such tetramers may comprise one or two, or three, or four ofany of AH3, e.g. SEQ ID NO: 59, AA6, e.g. SEQ ID NO: 60, A3H, e.g. SEQID NO: 61, AC1, e.g. SEQ ID NO: 62, A11G, e.g. SEQ ID NO: 63, and AE1,e.g. SEQ ID NO: 64, 2D, e.g. SEQ ID NO: 65, 2Ds, e.g. SEQ ID NO: 66, 5D,e.g. SEQ ID NO: 67, E3, e.g. SEQ ID NO: 68, 7F, e.g. SEQ ID NO: 69, 5E,e.g. SEQ ID NO: 70, B12, e.g. SEQ ID NO: 71, A11, e.g. SEQ ID NO: 72,AB8, e.g. SEQ ID NO: 73, C6, e.g. SEQ ID NO: 74, C12, e.g. SEQ ID NO:75, and A1, e.g. SEQ ID NO: 76, or variants thereof.

In certain embodiments of the composition, the binding protein isspecific for a toxin which is a Shiga toxin, and the binding region ofthe binding protein includes a recombinant camelid heavy-chain-onlyantibody having an amino acid sequence selected from the group:

(JET-A9, SEQ ID NO: 77)QVQLVETGGGLAQAGDSLRLSCVEPGRTLDMYAMGWIRQAPGEEREFVASISGVGGSPRYADSVKGRFTISKDNTKSTIWLQMNSLKPEDTAVYYCAAGGDIYYGGSPQWRGQGTRVTVSSEPKTPKPQ; (JGG-D4, SEQ ID NO: 78)QVQLVESGGGLVQAGGSLRLSCAASGRINGDYAMGWFRQAPGEEREFVAVNSWIGGSTYYTDSVKGRFTLSRDNAKNTLSLQMNSLKPEDTAVYYCAAGHYTDFPTYFKEYDYWGQGTQVTVSSEPKTPKPQ; (JEN-D10, SEQ ID NO: 79)QVQLVETGGLVQAGGSLRLSCAASGVPFSDYTMAWFRQAPGKEREVVARITWRGGGPYYGNSGNGRFAISRDIAKSMVYLHMDSLKPEDTAVYYCAASRLRPALASMASDYDYWGQGTQVSVSSEPKTPKPQ; (JGH-G1, SEQ ID NO: 80)QVQLVESGGGLVQPGESLRLSCVASASTFSTSLMGWVRQAPGKGLESVAEVRTTGGTFYAKSVAGRFTISRDNAKNTLYLQMNSLKAEDTGVYYCTAGAGPIATRYRGQGTQVTVSSAHHSEDP; (JEU-A6, SEQ ID NO: 81)QVQLVESGGGLVQPGGSLKLSCAASGFTLADYVTVWFRQAPGKSREGVSCISSSRGTPNYADSVKGRATVSRNNANNTVYLQMNGLKPDDTAIYYCAAIRPARLRAYRECLSSQAEYDYWGQGTQVTVSSAHHSEDP; (JEU-D2, SEQ ID NO: 82)QVQLVESGGGLVQPGGSLGLSCAMSGTTQDYSAVGWFRQAPGKEREGVSCISRSGRRTNYADSVRGRFTISRDNAKDTVYLQMNSLKPDDTAVYYCAARKTDMSDPYYVGCNGMDYWGKGTLVTVSSAHHSEDP; (JGH-G9, SEQ ID NO: 83)QVQLVESGGGLVQPGGSLTLSCTASGFTLNSYKIGWFRQAPGKEREGVSCINSGGNLRSVEGRFTISRDNTKNTVSLHMDSLKPEDTGVYHCAAAPALNVFSPCVLAPRYDYWGQGTQVTVSSAHHSEDP; (JFD-A4, SEQ ID NO: 84)QVQLVESGGGLVQPGGSLRLSCAASGFTLGSYHIGWFREIPPGKEREGTSCLSSRGDYTKYAEAVKGRFTISRDNTKSTVYLQMNNLKPEDTGIYVCAAIRPVLSDSHCTLAARYNYWGQGTQVTVSSAHHSEDP; (JFD-A5, SEQ ID NO: 85)QVQLVESGGGLVQPGGSLRLSCAALEFTLEDYAIAWFRQAPGKEREGVSCISKSGVTKYTDSVKGRFTVARDNAKSTVILQMNNLRPEDTAVYNCAAVRPVFVDSVCTLATRYTYWGEGTQVTVSSAHHSEDP; and (JGG-G6, SEQ ID NO: 86)QVQLVETGGGLVQPGGSLKLSCAASEFTLDDYHIGWFRQAPGKEREGVSCINKRGDYINYKDSVKGRFTISRDGAKSTVFLQMNNLRPEDTAVYYCAAVNPVFPDSRCTLATRYTHWGQGTQVTVSSAHHSEDP of variants thereof.

In certain embodiments amino acid sequence SEQ ID NO: 77 or a variantthereof, QVQLVETGGGLAQAGDSLRLSCVEPGRTLDMYAMGWIRQAPGEEREFVASISGVGGSPRYADSVKGRFTISKDNTKSTIWLQMNSLKPEDTAVYYCAAGGDIYYGGSPQWRGQGT RVTVSSEPKTPKPQ(JET-A9) binds to Stx1 or a portion or homolog thereof.

In certain embodiments amino acid sequence SEQ ID NO: 78 or a variantthereof, QVQLVESGGGLVQAGGSLRLSCAASGRINGDYAMGWFRQAPGEEREFVAVNSWIGGSTYYTDSVKGRFTLSRDNAKNTLSLQMNSLKPEDTAVYYCAAGHYTDFPTYFKEYDYWGQGTQVTVSSEPKTPKPQ (JGG-D4) binds to Stx1 or a portion or homologthereof.

In certain embodiments amino acid sequence SEQ ID NO: 79 or a variantthereof, QVQLVETGGLVQAGGSLRLSCAASGVPFSDYTMAWFRQAPGKEREVVARITWRGGGPYYGNSGNGRFAISRDIAKSMVYLHMDSLKPEDTAVYYCAASRLRPALASMASDYDYWGQGTQVSVSSEPKTPKPQ (JEN-D10) binds to Stx2 or a portion or homologthereof.

In certain embodiments amino acid sequence SEQ ID NO: 80 or a variantthereof, QVQLVESGGGLVQPGESLRLSCVASASTFSTSLMGWVRQAPGKGLESVAEVRTTGGTFYAKSVAGRFTISRDNAKNTLYLQMNSLKAEDTGVYYCTAGAGPIATRYRGQGTQVTVS SAHHSEDP(JGH-G1) binds to Stx2 or a portion or homolog thereof.

In certain embodiments amino acid sequence SEQ ID NO: 81 or a variantthereof, QVQLVESGGGLVQPGGSLKLSCAASGFTLADYVTVWFRQAPGKSREGVSCISSSRGTPNYADSVKGRATVSRNNANNTVYLQMNGLKPDDTAIYYCAAIRPARLRAYRECLSSQAEYDYWGQGTQVTVSSAHHSEDP (JEU-A6) binds to Stx2 or a portion or homologthereof.

In certain embodiments amino acid sequence SEQ ID NO: 82,QVQLVESGGGLVQPGGSLGLSCAMSGTTQDYSAVGWFRQAPGKEREGVSCISRSGRRTNYADSVRGRFTISRDNAKDTVYLQMNSLKPDDTAVYYCAARKTDMSDPYYVGCNGMDYWGKGTLVTVSSAHHSEDP (JEU-D2) binds to Stx2 or a portion or homologthereof.

In certain embodiments amino acid sequence SEQ ID NO: 83 or a variantthereof, QVQLVESGGGLVQPGGSLTLSCTASGFTLNSYKIGWFRQAPGKEREGVSCINSGGNLRSVEGRFTISRDNTKNTVSLHMDSLKPEDTGVYHCAAAPALNVFSPCVLAPRYDYWGQGT QVTVSSAHHSEDP(JGH-G9) binds to Stx2 or a portion or homolog thereof.

In certain embodiments amino acid sequence SEQ ID NO: 84 or a variantthereof, QVQLVESGGGLVQPGGSLRLSCAASGFTLGSYHIGWFRHPPGKEREGTSCLSSRGDYTKYAEAVKGRFTISRDNTKSTVYLQMNNLKPEDTGIYVCAAIRPVLSDSHCTLAARYNYWGQGTQVTVSSAHHSEDP (JFD-A4) binds to Stx1, Stx2, or both Stx1 and Stx2.In various embodiments SEQ ID NO: 84 binds to at least one of Stx1,Stx2, or a portion or homolog thereof.

In certain embodiments amino acid sequence SEQ ID NO: 85 or a variantthereof, QVQLVESGGGLVQPGGSLRLSCAALEFTLEDYAIAWFRQAPGKEREGVSCISKSGVTKYTDSVKGRFTVARDNAKSTVILQMNNLRPEDTAVYNCAAVRPVFVDSVCTLATRYTYWGEGTQVTVSSAHHSEDP (JFD-A5) binds to Stx1, Stx2, or both Stx1 and Stx2.In various embodiments SEQ ID NO: 85 binds to at least one of Stx1,Stx2, or a portion or homolog thereof.

In certain embodiments amino acid sequence SEQ ID NO: 86 or a variantthereof, QVQLVETGGGLVQPGGSLKLSCAASEFTLDDYHIGWFRQAPGKEREGVSCINKRGDYINYKDSVKGRFTISRDGAKSTVFLQMNNLRPEDTAVYYCAAVNPVFPDSRCTLATRYTHWGQGTQVTVSSAHHSEDP (JGG-G6) binds to Stx1, Stx2, or both Stx1 and Stx2.In various embodiments SEQ ID NO: 86 binds to at least one of Stx1,Stx2, or a portion or homolog thereof.

In various embodiments, the amino acid sequence of the compositionfurther includes an amino acid analog, an amino acid derivative, or aconservative substitution of an amino acid residue. The binding proteinin various embodiments includes an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NOs: 56-87and 95 or variants thereof. In related embodiments, substantiallyidentical means that the amino acid sequence of the binding protein hasat least about 50% identity, at least about 60% identity, at least about65% identity, at least about 70% identity, at least about 75% identity,at least about 80% identity, at least about 85% identity, at least about90% identity, at least about 95% identity, at least about 97% identity,at least about 98% identity, or at least about 99% identity to the aminoacid sequence of SEQ ID NOs: 56-87 and 95. Alternatively, the bindingprotein is encoded by at least one nucleotide sequence or the proteinincludes amino acid sequence selected from the group of SEQ ID NOs: 1-87and 95, and substantially identical to any of these sequences.

The composition in various embodiments further includes the bindingprotein or a source of expression of the binding protein selected fromthe group of: a purified binding protein preparation; a nucleic acidvector with a gene encoding the binding protein; a viral vector encodingthe binding protein; and a naked nucleic acid encoding the bindingprotein which is expressed from the DNA. In related embodiments, theviral vector is derived from a genetically engineered genome of at leastone virus selected from: an adenovirus, an adeno-associated virus, aherpes virus, and a lentivirus.

In a related embodiment of the composition, the binding protein isheterodimeric. In various embodiments, the heterodimeric binding proteinincludes a first binding region and a second binding region. For examplethe first binding region and the second binding region include VHHs, andthe first binding region binds specifically to a C. difficile TcdA andthe second binding region binds specifically to a C. difficile TcdB. Invarious embodiments, homo- or hetero-tetramers are also provided (e.g.four binding regions which bind specifically to a C. difficile TcdA,four binding regions which bind specifically to C. difficile TcdB, threebinding regions which bind specifically to C. difficile TcdA and onebinding region which binds specifically to C. difficile TcdB, threebinding regions which bind specifically to C. difficile TcdB and onebinding region which binds specifically to C. difficile TcdA, and twobinding regions which bind specifically to C. difficile TcdA and twobinding regions which binds specifically to C. difficile TcdB).

An aspect of the invention provides a kit for treating a subject exposedto or at risk for exposure to a disease agent including: apharmaceutical composition for treating a subject at risk for exposureto or exposed to a disease agent, the pharmaceutical compositionincluding: at least one recombinant heteromultimeric neutralizingbinding protein comprising a plurality binding regions, such that thebinding regions are not identical, and each binding region specificallybinds a non-overlapping portion of the disease agent, such that thebinding protein neutralizes the disease agent, thereby treating thesubject for exposure to the disease agent; a container; and,instructions for use. In various embodiments, the instructions for useinclude instructions for a method for treating a subject at risk forexposure to or exposed to a disease agent using the pharmaceuticalcomposition.

In various embodiments of the kit, the binding protein is selected fromthe group of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂.

In a related embodiment of the kit, the binding protein includes alinker. In various embodiments, the linker includes at least oneselected from: a peptide, a protein, a sugar, or a nucleic acid. Forexample, the linker includes amino acid sequence GGGGS (SEQ ID NO: 54),or GGGGSGGGGSGGGGS (SEQ ID NO: 55), or a portion or variant thereof.Alternatively, the linker includes a single amino acid or a plurality ofamino acids.

In related embodiments of the kit, the disease agent for which thebinding protein and binding regions are specific is selected from: avirus, a cancer cell, a fungus, a bacterium, a parasite, and a productof one of those such as a pathogenic molecule, a protein, alipopolysaccharide, or a toxin. In related embodiments, the toxin forwhich the binding protein is specific is a Botulinum neurotoxinincluding a serotype selected from: A, B, C, D, E, F, and G. In variousembodiments of the kit, the toxin for which the binding protein isspecific is at least one selected from the group of: staphylococcalα-hemolysin, staphylococcal leukocidin, aerolysin cytotoxic enterotoxin,a cholera toxin, a Bacillus cereus hemolysis II toxin, a Helicobacterpylori vacuolating toxin, a Bacillus anthracisi toxin, a cholera toxin,an Escherichia coli serotype O157:H7 toxin, an Escherichia coli serotype0104:H7 toxin, a lipopolysaccharide endotoxin, a Shiga toxin, apertussis toxin, a Clostridium perfringens iota toxin, a Clostridiumspiroforme toxin, a Clostridium difficile toxin A, a Clostridiumdifficile toxin B, a Clostridium septicum a toxin, and a Clostridiumbotulinum C2 toxin. In certain embodiments, the binding regions of thebinding protein are specific to different classes of disease agents,e.g., each of the plurality of binding regions is different and isspecific for an agent from bacteria, virus, fungus, cancer, and apathogenic molecule. For example a binding region is specific for avirus and another binding region is specific for a bacterium.

In a related embodiment of the kit, the binding protein is specific fora toxin which is a C. botulinum toxin, and the binding region includes arecombinant camelid heavy-chain-only antibody, such that thepharmaceutical composition includes the binding protein that has anamino acid sequence selected from the group consisting of: SEQ ID NO:56, SEQ ID NO: 57, SEQ ID NO: 58, or a portion or variant thereof.

In a related embodiment of the kit, the binding region of the bindingprotein is specific for a toxin which is a C. botulinum toxin A, suchthat the binding region of the binding protein includes a recombinantcamelid heavy-chain-only antibody having an amino acid sequence selectedfrom the group of: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 87, SEQ ID NO: 95, anda portion or variant thereof.

In a related embodiment of the kit, the toxin for which the bindingprotein is specific is a C. difficile toxin B, and the binding region ofthe binding protein includes a recombinant camelid heavy-chain-onlyantibody having an amino acid sequence selected from: SEQ ID NO: 65, SEQID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO:75, SEQ ID NO: 76, SEQ ID NO: 87, SEQ ID NO: 95, and a portion orvariant thereof. In certain embodiments, the binding protein and/orbinding regions are encoded by a nucleotide sequence or the bindingprotein and/or regions include an amino acid sequence selected from thegroup of SEQ ID NOs: 1-87 and 95, or are substantially identical tothese sequences.

In a related embodiment, the binding protein is specific for a Shigatoxin, and the binding region of the binding protein includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from: SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, and SEQ ID NO: 86 or variants thereof.

An aspect of the invention provides a composition including at least oneamino acid sequence selected from the group of: SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 94,SEQ ID NO: 95 or a portion or variant thereof. The composition invarious embodiments includes an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NOs: 59-86.In related embodiments, substantially identical means an amino acidsequence that has at least 60% identity, at least 65% identity, at least70% identity, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity, at least about97% identity, at least about 98% identity, or at least 99% identity toan amino acid sequence of any of SEQ ID NOs: 56-87 and 95.

An aspect of the invention provides a method for treating a subject atrisk for exposure to or exposed to a plurality of disease agents, themethod including: contacting the subject with at least one recombinantheteromultimeric neutralizing binding protein including two or morebinding regions, such that the binding protein neutralizes at least two(plurality) of disease agents, thereby treating the subject for exposureto the plurality of disease agents.

In a related embodiment of the method, the at least two of the bindingregions are identical. Alternatively, the at least two binding regionsinclude at least two non-identical binding regions. In relatedembodiments of the method, the binding protein is at least one selectedfrom the group of: a heterodimer, a trimer, a tetramer, a pentamer, anda hexamer. In various embodiments, the tetramer includes a homodimer ofa heterodimer, for example a heterodimer of AH3 and AA6 as is shown inSEQ ID NO: 95.

In various embodiments, the plurality from which the exemplary diseaseagents are selected from a virus, a cancer cell, a fungus, a bacterium,a parasite and a product thereof such as a pathogenic molecule, aprotein, a lipopolysaccharide, or a toxin. For example the diseaseagents include toxins such as TcdA and TcdB.

In related embodiments of the method, the binding protein includes atleast one selected from the group of SEQ ID NOs: 56-87 and 95 or aportion or a homologue or variant thereof.

In related embodiments of the method, the binding protein is selectedfrom the group of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂. In a related embodiment of themethod, the binding protein includes a linker located between each ofthe multimeric components of the binding regions. In variousembodiments, the linker is at least one selected from the group of: apeptide, a protein, a sugar, or a nucleic acid. For example, the linkercomprises amino acid sequence GGGGS (SEQ ID NO: 54) or amino acidsequence GGGGSGGGGSGGGGS (SEQ ID NO: 55).

In a related embodiment, the method further includes prior tocontacting, engineering the binding protein using a dimerization agent.In a related embodiment, the dimerization agent includes amino acidsequence

(SEQ ID NO: 94) TSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC,or a portion or variant thereof.

In various embodiments of the method, the plurality of disease agents isat least two selected from the group of: Staphylococcal α-hemolysin,Staphylococcal leukocidin, aerolysin cytotoxic enterotoxin, a choleratoxin, Bacillus cereus hemolysis II, and Helicobacter pylori vacuolatingtoxin, Bacillus anthracis, cholera toxin, Escherichia coli serotypeO157:H7, Escherichia coli serotype 0104:H7, lipopolysaccharideendotoxin, Shiga toxin, pertussis toxin, Clostridium perfringens iotatoxin, Clostridium spiroforme toxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium septicum a toxin, andClostridium botulinum C2 toxin. In related embodiments of the method,the binding protein includes at least one selected from the group of:SEQ ID NOs: 56-87 and 95 or variants thereof.

Binding Agent

The binding agent or binding protein is in one embodiment, a moleculethat binds to a portion of a target molecule, disease agent, or diseaseagent target. The binding protein treats the subject by any or all ofseveral mechanisms, including promoting clearance, phagocytosis,neutralization, inhibition, and activation of the immune response. Theterm “binding agent” or “binding protein”, includes in addition tofull-length antibodies, molecules such as antibody fragments (e.g.,single chain antibodies, and VHHs), microproteins (also referred to ascysteine knot proteins or knottins), darpins, anticalins, adnectins,peptide mimetic molecules, aptamers, synthetic molecules, and refers toany composition that binds to a target and/or disease agent and elicitsan immune effector activity against the molecule target and/or diseaseagent. In certain embodiments, the binding protein is a recombinantmultimeric neutralizing binding protein including two or more bindingregions, such that the binding regions are not identical, and eachand/or disease agent. Alternatively, the binding protein includesbinding regions that bind specifically to different types of diseaseagents such as different types of pathogenic molecules such as bacteria,viruses, fungi, allergens, and toxins. For example, at least one bindingregion of the binding protein bind to a virus surface protein, and atleast one different binding regions binds to a bacterial toxin.

The multimeric neutralizing binding protein herein in certainembodiments includes one or a plurality of epitopic tags. In certainembodiments, the binding protein includes a linker that covalentlyconnects each binding region of the heterodimer. For example, the linkeris a single amino acid or a sequence of a plurality of amino acids thatdoes not affect or reduce the stability, orientation, binding,neutralization, and/or clearance characteristics of the binding regionsand binding protein. In certain embodiments, each binding region isspecific to a non-identical disease agent. For example, the bindingprotein in certain embodiments includes a binding region specific to abacterium or bacterial toxin, and at least one other binding region isspecific to a virus, fungus, allergen, or to a non-identical bacteriumor bacterial toxin. For example, a multimeric binding protein in certainembodiments has binding regions specific to a TcdA and to a TcdA or to aShiga toxin, or the respective binding regions are specific to each of aBotulinum toxin and a virus.

In certain embodiments, the binding protein neutralizes or inhibits themolecule target and/or disease agent for example by preventing thedisease agent entry into cells. In certain embodiments, the bindingprotein upon being administered to the subject neutralizes the toxinand/or triggers an antibody mediated effector activity in the subject.

The binding protein is in certain embodiments a monomer (e.g., a singleunit), or includes a covalently bound protein including a plurality ofmonomers such as for example a dimer, a trimer, a tetramer, a pentamer,an octamer, a 10-mer, a 15-mer, a 20-mer, or any multimer. In certainembodiments, the binding protein is a monomer and the binding proteinhas one binding region that binds to an epitope of the molecule targetand/or disease agent. Alternatively, the binding protein in certainembodiments has two or more connected or joined monomers each with abinding region and each binding to an epitope of a disease agent or to aplurality of epitopes of disease agents. The multimeric binding proteinin certain embodiments includes the same monomer. Alternatively, themultimeric binding protein includes monomers or binding regions or acombination thereof (i.e., heteromulteric). Accordingly, the multimerscan be homogeneous such that each includes two or more monomers having abinding region that binds to the same site of a disease agent.Alternatively, the multimers are heterogeneous and include two or moremonomers having a binding region that binds to two or more differentsites of one or more disease agents. The heterogeneous multimers(heteromultimers) bind non-overlapping portions of the molecule targetand/or disease agent. In various embodiments, the binding protein is ahomodimer of a heterodimer or a heterotrimer. In a related embodiment,the heteromultimers bind a plurality of non-identical epitopes on aplurality of disease agents.

In certain embodiments the binding protein includes a single tag,multiple tags, for example each multimeric binding protein includes twoor more tags on each component binding region (i.e., monomer).Alternatively, the heterodimer comprises no tag attached to the monomersand/or linker. In certain embodiments, presence of the tag on oroperably fused to the binding protein and/or binding regionsynergistically induces clearance of the disease agent from the body.For example, the tag attached to the binding protein induces an immuneresponse from a patient or subject contacted with a pharmaceuticalcomposition containing the tagged-binding protein. In certainembodiments the tag includes a portion (e.g., conserved, unique,inactivated, and non-functional) of a pathogenic molecule. In certainembodiments, the tag is an adjuvant. See Gerber et al. U.S. Pat. No.7,879,333 issued Feb. 1, 2011 which is incorporated by reference hereinin its entirety. For example, the tag is a peptide, carbohydrate,polymer, or nucleic acid that is effective for enhancing neutralizationand/or clearance of the disease agent or plurality of disease agents.

The multimeric binding protein in certain embodiments is a heterodimerhaving two tags, one tag attached to each monomer, or alternatively theheterodimer includes one tag on each monomer or one tag total on one ofthe two monomers. The term “heterodimer” includes a single proteinhaving two different monomers are joined by a linker. Data herein shownthat a heterodimers having two E-tags effectively protected animalsexposed to hundreds-fold and/or thousands-fold the lethal dose of asingle disease agent such as a C. difficile toxin A. Examples hereinshow that recombinant multimeric binding proteins, having two or morenon-identical binding regions, administered to subjects either before orafter contact with a disease agent resulted in comparable and betterantitoxin efficacy than serum-based polyclonal antitoxins.

The binding agents/proteins described herein include bindingagent/protein portions, regions, and fragments. For example, the bindingprotein is an antibody and, in certain embodiments the binding proteinincludes antibody fragments. The term “antibody fragment” refers toportion of an immunoglobulin having specificity to a molecule targetand/or disease agent, or a molecule involved in the interaction orbinding of the molecule target and/or disease agent. The term “antibodyfragment” encompasses fragments from binding protein, for example bothpolyclonal and monoclonal antibodies including transgenically producedantibodies, single-chain antibodies (scFvs), recombinant Fabs, andrecombinant heavy-chain-only antibodies (VHHs), e.g., from any organismproducing VHH antibody such as a camelid, a shark, or a designed VHH.

VHHs are antibody-derived therapeutic proteins that contain the uniquestructural and functional properties of naturally-occurring heavy-chainantibodies. VHH technology is based on fully functional antibodies fromcamelids that lack light chains. These heavy-chain antibodies contain asingle variable domain (VHH) and two constant domains (CH2 and CH3). Thecloned and isolated VHH domain is a stable polypeptide harboring theantigen-binding capacity of the original heavy-chain antibody. SeeCastorman et al. U.S. Pat. No. 5,840,526 issued Nov. 24, 1998; andCastorman et al. U.S. Pat. No. 6,015,695 issued Jan. 18, 2000, each ofwhich is incorporated by reference herein in its entirety. VHHs arecommercially available from Ablynx Inc. (Ghent, Belgium) under thetrademark of NANOBODIES™.

Suitable methods of producing or isolating antibody fragments having therequisite binding specificity and affinity are described herein andinclude for example, methods which select recombinant antibody from alibrary, by PCR (See Ladner U.S. Pat. No. 5,455,030 issued Oct. 3, 1995and Devy et al. U.S. Pat. No. 7,745,587 issued Jun. 29, 2010, each ofwhich is incorporated by reference herein in its entirety).

Functional fragments of antibodies, including fragments of chimeric,humanized, primatized, veneered or single chain antibodies, can also beproduced. Functional fragments or portions of the foregoing antibodiesinclude those which are reactive with the disease agent. For example,antibody fragments capable of binding to the disease agent or portionthereof, including, but not limited to scFvs, Fabs, VHHs, Fv, Fab, Fab′and F(ab′)2 are encompassed by the invention. Such fragments can beproduced by enzymatic cleavage or by recombinant techniques. Forinstance, papain or pepsin cleavage are used generate Fab or F(ab′)₂fragments, respectively. Antibody fragments are produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain peptide portion can bedesigned to include DNA sequences encoding the CH₁ peptide domain andhinge region of the heavy chain. Accordingly, the present inventionencompasses a polynucleic acid that encodes the binding proteindescribed herein (e.g., a binding fragment with a tag). Binding proteinsin certain embodiments are made as part of a multimeric protein, themonomer or single binding region (e.g., antibody fragments,microproteins, darpins, anticalins, adnectins, peptide mimeticmolecules, aptamers, synthetic molecules, etc) can be linked. Anycombination of binding protein or binding region types can be linked. Inan embodiment, the monomer or binding region of a multimeric bindingprotein can be linked covalently. In another embodiment, a monomerbinding protein can be modified, for example, by attachment (directly orindirectly (e.g., via a linker or spacer)) to another monomer bindingprotein. A monomer in various embodiments is attached or geneticallyfused to another monomer e.g., by recombinant protein that is engineeredto contain extra amino acid sequences that constitute the monomers.Thus, the DNA encoding one monomer is joined (in reading frame) with theDNA encoding the second monomer, and so on. Additional amino acids incertain embodiments are encoded between the monomers that produce anunstructured region separating the different monomers to better promotethe independent folding of each monomer into its active conformation orshape. Commercially available techniques for fusing proteins are used invarious embodiments to join the monomers into a multimeric bindingprotein of the present invention.

The term “antagonist” as used herein includes proteins or polypeptidesthat bind to the disease agent, inhibit function of the disease agent,and are included in certain embodiments to the binding region of thebinding protein.

A binding protein includes any amino acid sequence that binds to thedisease agent or target including molecules that have scaffolds.Examples of binding proteins having scaffolds are DARPins, Anticalins,and AdNectins. DARPins are derived from natural ankyrin repeat proteinsand bind to proteins including e.g., human receptors, cytokines,kinases, human proteases, viruses and membrane proteins (MolecularPartners AG Zurich Switzerland). Anticalins are derived from lipocalins,and comprise a hypervariable loops supported by a conserved β-sheetframework, which acts as a binding protein. (Pieris AG, Germany). Thescaffold for anticalins are lipocalins. AdNectins are derived from humanfibronectin (e.g., the scaffold), and bind to targets of various medicalconditions and are commercially available from Adnexus (Waltham, Mass.).See also Alexandru et al. U.S. Pat. No. 7,867,724 issued Jan. 11, 2011,which is incorporated by reference herein in its entirety. In certainembodiments, the binding protein having the scaffold is encoded by anucleotide sequence or the binding protein includes an amino acidsequence that is substantially identical or homologous to the sequencesdescribed herein, for example SEQ ID NO: 1-87 and 95 or variantsthereof. Recombinant multimeric binding proteins herein include aminoacid sequences from a binding protein sequence having conservativesequence modifications. As used herein, the term “conservative sequencemodifications” refers to amino acid modifications that do notsignificantly affect or alter the characteristics (e.g., neutralization,clearance, binding, stability, and orientation) of the binding protein,i.e., amino acid sequences of binding protein that present these sidechains at the same relative positions will function in a manner similarto the binding protein. Such conservative modifications include aminoacid substitutions, additions and deletions. Modification of the aminoacid sequence of recombinant multimeric binding protein is achievedusing any known technique in the art e.g., site-directed mutagenesis orPCR based mutagenesis. Such techniques are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1989. Conservative aminoacid substitutions are modifications in which the amino acid residue isreplaced with an amino acid residue having a similar side chain such asreplacing a small amino acid with a different small amino acid, ahydrophilic amino acid with a different hydrophilic amino acid, etc.

Examples herein show that a molecule target and/or disease agent isbound by a binding protein, the molecule target and/or disease agentexemplified by a bacterial toxin released by the pathogen, for example abotulinum toxin. Botulinum toxin serotypes A to G are synthesized byorganisms including Clostridium botulinum, Clostridium baratii, andClostridium butyricum. Simpson, L. L 2004 Annu. Rev. Pharmacol. Toxicol.44: 167-193. C. botulinum produces serotypes A to G, C. baratii producesserotype F, and C. butyricum produces serotype E only. The structuresand substrates for each of the botulism toxin serotypes as well as theserotype specific cleavage sites have been determined, and the mechanismof toxin killing has been elucidated. The botulinum toxin actspreferentially on peripheral cholinergic nerve endings to blockacetylcholine release, and causes disease (i.e., botulism) and can beused to treat disease (e.g., dystonia). Ibid., Abstract. Thetoxigenicity of botulinum toxin depends on penetration of the toxinthrough cellular and intracellular membranes. Thus, toxin that isingested or inhaled binds to epithelial cells and is transported to thegeneral vascular circulation. Toxin that reaches peripheral nerveendings binds to the cell surface then penetrates the plasma membrane byreceptor-mediated endocytosis and the endosome membrane by pH-inducedtranslocation. Ibid., Abstract. Internalized toxin acts in the cytosolas a metalloendoprotease to cleave polypeptides that are essential forexocytosis.

Examples herein show binding proteins/agents that specifically bind eachof a variety of distinct serotypes of a microbial neurotoxin that causesbotulism, BoNT/A and BoNT/B. The amino acid sequence of the bindingagents include scFvs and VHHs for example SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52 or combinations or portions or variants thereof. The correspondingnucleic acid sequences of binding agents are shown in SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51 or a combination or variant thereof. In various embodimentsthe amino acid sequence of the binding agents includes VHHs for exampleSEQ ID NO: 56-87 or 95 or combinations or portions or variants thereof.In certain embodiments, the binding agent includes a tag that wasengineered as a portion of the binding agent, for example the tag hasamino acid sequence of SEQ ID NO: 15, and is genetically fused to thecarboxyl end of the binding agents. In certain embodiments, the tagenhances ability of the binding protein to neutralize and/or clear thedisease agent from the subject. FIG. 5 shows a phylogenetic tree ofJDQ-B5 (SEQ ID NO: 24), a VHH binding agent that specifically binds toBoNT/A and other VHHs that compete with JDQ-B5 for binding to BoNT/A.The length of the branches in the tree represents the relatedness of thesequences with the shorter branches indicating greater relatedness(i.e., homology) and the longer branches indicating less homology of theamino acid sequences.

The present invention provides a number of different binding proteins,each having binding regions with specificity and affinity to targetdifferent areas of one or more disease agents. In an embodiment, two orthree binding proteins specific to different epitopes of a disease agentare used. In a disease having a number of disease agents involved incausing the disease or condition, such as botulism, multiple diseaseagents are targeted by the compositions and methods herein. In the caseof botulism, since any one of at least seven neurotoxin serotypes areinvolved, a pool/mixture of binding proteins is prepared containingbinding proteins for a plurality of known serotypes that cause thedisease in humans. Botulism is often caused by exposure to a single BoNTserotype, and it is generally difficult to quickly determine whichserotype is the cause. Thus, the standard of care in treating botulismincludes administration of a number of antibodies to protect againstmost if not all of the serotypes that cause the disease in human. Hence,it is appropriate to protect subjects from botulism, to stockpilebinding proteins that bind to several or preferably all known serotypesthat cause botulism.

The present invention in various embodiments further encompassescompositions that are multimeric binding proteins having two or moremonomers in which a monomer is exemplified by a VHH amino sequenceherein. In various embodiments, the composition includes at least oneselected from the group of SEQ ID NOs: 56-87 and 95 or variants thereof.Compositions further include nucleic acid sequences that encode theamino acids sequences herein, for example SEQ ID NO: 56-87 and 95 orvariants thereof. In certain embodiments, the monomer or binding regionincludes at least one sequence described herein, for example SEQ ID NOs:1-87 and 95 or variants thereof. An embodiment of a multimeric bindingprotein includes two or more of the VHH sequences herein expressed as asingle protein. Any combination of two or more of the VHH sequencesforms a multimeric binding protein of the present invention. In aparticular embodiment, the present invention relates to a heterodimer,i.e., protein, in which any two different VHH sequences herein areexpressed as a single protein, i.e., linked and expressed as a geneticfusion.

The binding protein in certain embodiments is a multimeric fusionprotein engineered and produced using a multimerization agent to form acomplex that effectively binds to and neutralizes a disease agent orplurality of disease agents (Shoemaker et al. U.S. publication number20130058962 published Mar. 7, 2013, which is incorporated by referenceherein in its entirety). In certain embodiments, the multimerizationagent includes a dimerization sequence for example including an aminoacid sequence shown in SEQ ID NO: 94. For example the dimerzation agentcomplexes peptide fragments each containing at least: about five to 25amino acids, about 25 to 50 amino acids, about 50 to 100 amino acids,about 100 to 150 amino acids, and about 150 amino acids to about 200amino acids. Multimerization agents and methods of using the agents forforming multimeric binding proteins are shown herein in Example 21. Seealso Moore et al. U.S. Pat. No. 7,763,445 issued Jul. 24, 2012 andCarter et al. U.S. Pat. No. 8,216,865 issued Jul. 10, 2012, each ofwhich is incorporated by reference herein in its entirety.

The disease agent target is any from different classes of pathogens,infectious agents or other unwanted material. A multi-target approach iswithin the scope of the methods and compositions herein, exemplified bya binding protein that binds to a viral disease agent, a bacterialdisease agent, a parasite disease agent, a cancer cell, and a proteinproduced therefrom and any combination thereof. In various embodiments,a binding protein neutralizes a plurality of pathogens or unwantedmaterial. Examples herein show a VHH heterodimer that binds to andneutralizes both TcdA and TcdB.

The disease agent, pathogen or infectious agent that is neutralized bythe binding agent is any molecule, virus or bacterium that infects amammal (e.g., human, horse, dog, goat, and cow) or a mammalian cell. Incertain embodiments, the disease agent is a bacterium selected fromActinobacillus, Bacillus, Borrelia, Brucella, Campylobacter, Chlamydia,Clostridium, Coxiella, Enterococcus, Escherichia, Francisella,Hemophilus, Legionella, Mycobacterium, Neisseria, Pasteurella,Pneumophila, Pseudomonas, Rickettsia, Salmonella, Shigella,Staphylococcus, Streptococcus, Treponema, and Yersinia. Alternatively,the disease agent is a virus including for example humanimmunodeficiency virus, foot-and-mouth disease virus, avian influenzavirus, and sheep pox virus.

The binding agent in various embodiments binds to and neutralizes aninfectious agent and/or a disease agent associated with a pathologyresulting from overexpression of a self protein in the subject such asan immunoglobulin, a leukocyte, a cytokine, and a growth factor. Forexample, the overexpression is of an inflammatory agent such as a tumornecrosis factor (e.g., TnFα) or an interleukin (IL) such as IL-1 beta,or IL-6. Alternatively, an infectious agent and/or a disease agent isassociated with expression of a mutated or modified molecule such as aprotein, a sugar, a glycoprotein, or expression of a cell carrying anucleotide sequence encoding the disease agent.

The binding agent in various embodiments binds to a cancer cell and/orcancer marker. For example, the cancer cell includes a melanoma; acarcinoma (e.g., colon carcinoma); a pancreatic cancer; a sarcoma; alymphoma; a leukemia; a brain tumor such as glioma; a lung cancer; anesophageal cancer; a mammary (breast) cancer; a bladder cancer; aprostate cancer; a head and neck cancer; an ovarian cancer; a kidneycancer; or a liver cancer.

The binding agents described herein are used in certain embodiments totreat symptoms of an autoimmune disease, a class of disorder whichincludes Hashimoto's thyroiditis; idiopathic myxedema, a severehypothyroidism; multiple sclerosis, a demyelinating disease marked bypatches or hardened tissue in the brain or the spinal cord; myastheniagravis which is a disease having progressive weakness of muscles causedby autoimmune attack on acetylcholine receptors at neuromuscularjunctions; Guillain-Barre syndrome, a polyneuritis; systemic lupuserythematosis; uveitis; autoimmune oophoritis; chronic immunethrombocytopenic purpura; colitis; diabetes; Grave's disease, which is aform of hypothyroidism; psoriasis; pemphigus vulgaris; and rheumatoidarthritis (RA).

It will be appreciated that in certain embodiments, the binding agent(e.g., peptide, protein, or portion or homolog thereof) of thisinvention can be obtained from a peptide synthesizer or any commercialsupplier of custom peptides produced synthetically, e.g., by solid phaseprocedures. For example, peptide synthesis can be performed usingvarious solid-phase techniques (Roberge et al. 995 Science 269:202) andautomated synthesis may be achieved, for example, using the 431A peptidesynthesizer (available from Applied Biosystems of Foster City, Calif.)in accordance with the instructions provided by the manufacturer. Seealso Horowitz et al. U.S. Pat. No. 8,131,480 issued Mar. 6, 2012.

Molecule Target and Disease Agent Target

A molecule target and/or disease agent target is any target which isbiological (e.g., protein, sugar, carbohydrate, DNA, RNA) or chemical towhich the binding protein binds, and is any target associated with adisease, defect or negative condition. The molecule target or diseaseagent target is any molecule capable of being bound, or whose activityis altered (e.g., neutralized, reduced or ceased), or that can berecognized by immune effectors and leads for example to clearance,opsonization, killing, and phagocytosis. For example, the disease agenttarget in certain embodiments is a portion of a pathogen or a moleculereleased or secreted by the pathogen (e.g. toxin). A pathogen is anagent that causes a disease or condition, and includes a virus, cancercell, bacterium, parasite or pathogenic protein. The disease agenttarget includes a pathogenic protein that is derived from normal cells,such as prions. The pathogenic protein or other molecule that is diseaseagent target is either independent of the pathogen or is associated withor produced by the pathogen.

In certain embodiments, the disease agent is a molecule (e.g, peptide)that is naturally produced by a plant or bacterium that inactivates ordisrupts normal function of cellular membranes, cellular compartments,or cellular organelles. For example, the disease agent disrupts functionof ribosomes.

A virus is a microscopic particle that infects the cells of a biologicalorganism and replicates in the host cell. In various embodiments, viralantigens including viral proteins, are targeted by the binding protein.Binding proteins bind to molecules or receptors on the virus, and areneutralized and/or cleared using the methods described herein. Examplesof viruses that are neutralized and/or cleared by the binding proteinherein include Influenza, Rhinovirus, Rubeola, Rubella, Herpes,Smallpox, Chickenpox, Human Papilloma, Rabies, and HumanImmunodeficiency viruses.

A parasite is an organism that lives on or in a different organism.Parasites have or express molecules that are used as a target by thebinding agent. Types of parasites include endoparasites (e.g., parasitesthat live inside the body of the host) and ectoparasites (e.g.,parasites that live on the outside of the host's body). Examples ofparasites that are treated by the methods, compositions, and kits hereinare shown in Horvitz et al. U.S. patent publication 20110010782published Jan. 13, 2011. Exemplary parasites include a protozoan (e.g.,a plasmodium, a cryptosporidium, a microsporidium, and isospora), atick, a louse and a parasitic worm.

Molecules on cancer cells also are targets of the binding agent. Inrelated embodiments, the target is a protein on the cancer cell such asa cancer marker. Examples of proteins or receptors associated withcancer cells include CD33, HER2/neu, CA 125 (MUC16), prostate-specificantigen (PSA), and CD44.

The disease agent target in certain embodiments includes bacteriaincluding Gram negative and Gram positive bacteria. Examples ofpathogenic bacteria bound by the binding protein include Clostridium,Staphylococcus, Neisseria, Streptococcus, Moraxella, Listeria, any ofthe Enterobacteriaceae, Escherichia coli, Corynebacterium, Klebsiella,Salmonella, Shigella, Proteus, Pseudomonas, Haemophilus, Bordetella,Legionella, Campylobacter, Helicobacter, and Bacteroides. For example,the disease agent target is Bacillus anthracis (Decker, J. 2003 DeadlyDiseases and Epidemics, Anthrax. Chelesa House Publishers. pages 1-112).

Enterohemorrhagic Escherichia coli (EHEC) is an emerging food- andwater-borne pathogen that colonizes the distal ileum and colon andproduces potent cytotoxins (Donnenberg, “Infections due to Escherichiacoli and other enteric gram-negative bacilli,” in ACP Medicine, WebMDProfessional Publishing, Danbury Conn., Chapter 7, pp. 8-1 to 8-18,2005). After ingestion of contaminated food, humans develop symptomsranging from mild diarrhea to the severe, and at times life-threatening,hemolytic uremic syndrome (HUS). Currently, EHEC is the most commoncause of pediatric renal failure in the United States (Mead et al, EmergInfect Dis, 5:607-625, 1999). Several EHEC serotypes cause disease, butthe 0157 serotype is by far the most common cause of EHEC-relateddisease in North America, Europe and Japan (Feng, “Escherichia coli” inGarcia (ed.) Guide to Foodborne Pathogens. John Wiley and Sons, Inc.,pp. 143-162, 2001). See also Waldor et al., U.S. patent publicationnumber 2010/0092511 A1 published Apr. 15, 2012, which is incorporated byreference herein in its entirety.

Shiga toxins are a family of related toxins with two major groups, Stx1and Stx2 (Friedman et al., 2001 Curr Opin Microbiol 4 (2): 201-7). Thetoxins are named for Kiyoshi Shiga, who first described the bacterialorigin of dysentery caused by Shigella dysenteriae. The most commonsources for Shiga toxin are the bacteria S. dysenteriae and theShigatoxigenic group of Escherichia coli (STEC), which includesserotypesO157:H7, 0104:H4, and other enterohemorrhagic E. coli, EHEC (Spears etal. 2006 FEMS Microbiology Letter 187-202; Sandvig et al. 2000 EMBO J 19(22): 5943-5950; and Krautz-Peterson et al. 2008 Infection and Immunity76(5) 1931-1939; and Vermeij U.S. Pat. No. 7,807,184 issued Oct. 5,2010, each of which is incorporated by reference herein in its entirety.Symptoms associated with Shiga toxin-exposure caused infection by EHECinclude watery stool followed by severe abdominal pain and bloody stool.Exposed persons develop complications leading to HUS, encephalopathy,and even death (Masuda et al., U.S. Pat. No. 7,345,161 issued Mar. 18,2008).

Methods for ascertaining the target molecule or disease agent aredescribed herein and depend on the type of molecule being inhibited. Forexample, in a case in which a class or group of bacteria are to beinhibited, conserved regions of bacteria are targeted, and bindingagents that bind to these targets are constructed. Methods for targetinga conserved region or polymorphic region of a nucleotide sequence thatencodes the target molecule, or the target molecule having an amino acidsequence are shown in Cicciarelli et al., U.S. patent publication number2005/0287129 A1 published Dec. 29, 2005 which is incorporated byreference herein in its entirety. In other embodiments, if a specificdisease agent such as a bacterium is to be inhibited, a non-conservedregion of the disease agent is targeted with the binding agents. Thebinding of the agents are determined and/or measured for example usingstandard assays, for example an enzyme-linked immunosorbent assay(ELISA), western blot and radioimmunoassay.

A molecule target or a disease agent target includes pathogenicmolecules including polypeptides or toxins to which the binding proteindescribed herein binds, neutralizes and/or clears. The term “pathogenicprotein” refers to a protein that can cause, directly or indirectly, adisease, or condition in an individual. A pathogenic protein is forexample a protein or a toxin produced by a bacterium, a virus, or acancer cell. A recombinant multimeric binding protein described hereinbinds non-overlapping areas of the disease agent target (e.g., a toxinproduced by a bacterium) and protects the subject from the pathology ofthe disease agent target by neutralizing and/or clearing the target. Thebinding protein protects subjects from negative symptoms caused byexposure to the disease agent target, and the risk of negative symptomscaused by a potential exposure to the target.

Anti-tag antibody described herein is used in various embodiments toeffect or facilitate effector functions. The anti-tag antibody includesfor example an immunoglobulin such as IgA, IgD, IgE, IgG, and IgM, andsubtypes thereof. In addition to monoclonal antibodies, polyclonalantibodies specific to the tag are used in the methods, compositions andkits described herein. Effector functions are performed for exampleimmune molecules interaction with the Fc portion of the immunoglobulin.Depending on the type of immunoglobulin chosen, the effector functionsresults in clearance of the disease agent (e.g., excretion, degradation,lysis or phagocytosis).

Mammalian antibody types IgA, IgD, IgE, IgG, and IgM, and antibodysubtypes are classified according to differences in their heavy chainconstant domains. Each immunoglobulin class differs in its biologicalproperties and characteristics. IgA is found for example in areascontaining mucus (e.g. in the gut, respiratory tract, and urogenitaltract) and prevents the colonization of mucosal areas by pathogens. IgDfunctions as a disease agent receptor on B cells. IgE binds to allergensand triggers histamine release from mast cells and also providesprotection against helminths (worms). IgG, in four forms, provides themajority of antibody-based immunity against invading pathogens. IgM hasa very high affinity for eliminating pathogens in the early stages of Bcell mediated immunity, and is expressed on the surface of B cells andalso in a secreted form.

Leukocytes such as mast cells and phagocytes have specific receptors onthe cell surface for binding antibodies. These Fc receptors interactwith the Fc region of classes of antibodies (e.g. IgA, IgG, IgE). Theengagement of a particular antibody with the Fc receptor on a particularcell triggers the effector function of that cell. For example,phagocytes function to perform phagocytosis, and mast cells function todegranulate. Effector functions generally result in destruction of aninvading microbe. In various embodiments, the type of immunoglobulin ischosen specifically for a type of desired effector function.

The present invention includes methods of administering one or morerecombinant multimeric binding proteins to a subject (e.g., human, cow,horse, pig, mouse, dog, and cat). The binding protein is administered incertain embodiments as a monomer, or as a multimeric binding proteincomprising a plurality of monomers having different binding regions. Themethods and compositions herein involve administration of one or moremultimeric binding agents that include monomers that each has a bindingregion that is specific to the disease agent. The binding agent forexample includes one or more tags. The binding agent/protein binds tothe target region on the disease protein. Administration of two or morebinding proteins (e.g., monomer binding proteins or multimeric bindingproteins), in various embodiments, increased the effectiveness of theantibody therapy, and reduced the severity of one or more negativesymptoms of exposure of the disease protein target. The binding proteinis administered in various embodiments as a single monomer, a mixture ofmultiple (e.g., two or more) monomers, a multimeric binding proteinincluding a plurality of monomers that are same or different, a mixtureof multiple (e.g., two or more) multimeric binding proteins comprisingmore than one monomer, or any combination thereof. Examples herein showthat administration of a binding protein containing more than one copyof the tag resulted in increased protection against a disease agenttarget, e.g., botulinum toxin serotype A. A single anti-tag antibodytype in certain embodiments binds to all binding proteins having a tag.In certain embodiments in which the binding proteins have multiplecopies (e.g., two or more) of the same tag, the anti-tag antibody bindsto each copy of the tag on the binding protein. The phrase, “antibodytherapeutic proteins” or “antibody therapeutic preparation” refers toone or more compositions that include at least one binding protein andoptionally at least one anti-tag antibody. The multimeric bindingprotein preparation in certain embodiments contains additional elementsincluding carriers as described herein.

The administration of the one or more binding proteins and/or anti-tagantibody is performed in related embodiments simultaneously orsequentially in time. The binding protein in certain embodiments isadministered before, after or at the same time as another bindingprotein or the anti-tag antibody, providing that the binding proteinsand/or the anti-tag antibodies are administered close enough in time tohave the desired effect (e.g., before the binding proteins have beencleared by the body). Thus, the term “co-administration” is used hereinto mean that the binding proteins and another binding protein or theanti-tag antibody are administered at time points to achieve effectivetreatment of the disease, and reduction in the level of the pathogen(e.g., virus, bacteria, cancer cell, proteins associated therewith, orcombination thereof) and symptoms associated with it. The methods of thepresent invention are not limited by the amount of time in between whichthe binding proteins and/or anti-tag antibody are administered;providing that the compositions are administered close enough in time toproduce the desired effect. In certain embodiment, the binding proteinsis administered only, alternatively the binding protein and/or anti-tagantibody are premixed and administered together. The binding proteinsand/or anti-tag antibody are in certain embodiments co-administered withother medications or compositions suitable to treating the diseaseagent.

The binding protein in certain embodiments is administered prior to thepotential risk of exposure to the disease target agent to protect thesubjects from symptoms of the disease agent target. For example, thebinding protein and/or clearing antibody is administered minutes, hoursor days prior to the risk of exposure. Alternatively, the bindingprotein is administered contemporaneously to the risk of exposure to thedisease agent target, or slightly after the risk of exposure. Forexample, the binding protein is administered to a subject at the momentthe subjects contacts, enters or passes through an environment (e.g.,room, hallway, building, and field) containing the risk of exposure tothe disease agent.

The methods of the present invention include treating a bacterialdisease, a parasitic infection, a viral disease, a cancer, smallunwanted molecule, a protein or a toxin associated therewith. This isaccomplished by administering the binding proteins and anti-tagantibodies described herein to the affected individual or individual atrisk. Administration ameliorates or reduces the severity of one or morethe symptoms of the disease or condition. The presence, absence orseverity of symptoms is measured for example using tests and diagnosticprocedures known in the art. Presence, absence and/or level of thedisease agent are measured in certain embodiments using methods known inthe art. Symptoms or levels of the disease agent can be measured at oneor more time points (e.g., before, during and after treatment, or anycombination thereof) during the course of treatment to determine if thetreatment is effective. A decrease or no change in the level of thedisease agent, or severity of symptoms associated therewith indicatesthat treatment is working, and an increase in the level of the diseaseagent, or severity of symptoms indicates that treatment is not working.Symptoms and levels of disease agents are measured in variousembodiments using methods known in the art. Symptoms that are monitoredin certain embodiments include fever, plain including headache, jointpain, muscular pain, difficulty breathing, lethargy, and impairedmobility, appetite and unresponsiveness. Toxin protection is assessed asincreased survival and reduction or prevention of symptoms. Methods,compositions and kits using the binding protein decrease and alleviatethe symptoms of the disease target agent and also improve survival fromexposure to the agent.

The antibody therapeutic agents including one or more binding proteinsor agents, and/or an anti-tag antibody are administered in variousembodiments with one or more pharmaceutical carriers. The terms“pharmaceutically acceptable carrier” and a “carrier” refer to anygenerally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. The binding agents and anti-tag antibodyare administered with or without a carrier. Exemplary carriers includecalcium carbonate, sucrose, dextrose, mannose, albumin, starch,cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, riceflour, magnesium stearate, and the like. Suitable formulations andadditional carriers are described in Remington's PharmaceuticalSciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of whichare incorporated herein by reference in their entirety. The bindingagents and anti-tag antibody are administered systemically or locally(e.g., by injection or diffusion).

Suitable carriers (e.g., pharmaceutical carriers) include, but are notlimited to sterile water, salt solutions (such as Ringer's solution),alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. The binding protein preparations are sterilized and, ifdesired, mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the active compounds. The bindingprotein preparations in certain embodiments are combined where desiredwith other active substances, e.g., enzyme inhibitors, to reducemetabolic degradation. A carrier (e.g., a pharmaceutically acceptablecarrier) is used optionally in certain embodiments to administer one ormore binding agents and an anti-tag antibody.

The binding agents and anti-tag antibodies in certain embodiments areadministered topically (as by powders, ointments, or drops), orally,rectally, mucosally, sublingually, parenterally, intracisternally,intravaginally, intraperitoneally, bucally, ocularly, or intranasally,depending on preventive or therapeutic objectives and the severity andnature of a exposure or risk of exposure to the disease agent target.The composition in various embodiments is administered in a single doseor in more than one dose over a period of time to confer the desiredeffect.

An effective amount of compositions of the present invention variesaccording to choice of the binding agent, the particular compositionformulated, the mode of administration and the age, weight and conditionof the patient, for example. As used herein, an effective amount of thebinding agents and/or anti-tag antibody is an amount which is capable ofreducing one or more symptoms of the disease or conditions caused by themolecule target or disease agent target. Dosages for a particularpatient are determined by one of ordinary skill in the art usingconventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol).

A composition in certain embodiments includes one or more nucleotidesequences described herein that encode the binding protein. In variousembodiments, a nucleotide sequence is either present as a mixture or inthe form of a DNA molecule a multimer. In various embodiments, thecomposition includes a plurality of nucleotide sequences each encodingthe binding protein including a monomer or polypeptide, or anycombination of molecules described herein, such that the binding proteinis generated in situ. In such compositions, a nucleotide sequence isadministered using any of a variety of delivery systems known to thoseof ordinary skill in the art, including nucleic acid expression systems,bacterial and viral expression systems. Appropriate nucleic acidexpression systems contain appropriate nucleotide sequences operablylinked for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve administrationof a bacterium (such as Bacillus-Calmette-Guerrin) that expresses thepolypeptide on its cell surface. In an embodiment, the DNA can beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which uses a non-pathogenic(defective), replication competent virus. Techniques for incorporatingDNA into such expression systems are well known to those of ordinaryskill in the art. The DNA can also be “naked,” as described, forexample, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed byCohen, Science 259:1691-1692, 1993. The uptake of naked DNA can beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into recipient cells.

Systems or kits of the present invention include in various embodimentsone or more binding agents having a binding region and one or more tags,and an anti-tag antibody having an anti-tag region (e.g., an anti-tagantibody), as described herein.

The methods, compositions and kits described herein in certainembodiments include isolated polypeptide molecules that have beenengineered or isolated to act as binding agents or binding proteins. Abinding protein composition includes for example an amino acid sequenceselected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or combinations thereof.In various embodiments, a binding protein composition includes anucleotide sequence that encodes an amino acid sequence, for example thenucleotide sequence is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 orcombinations thereof. The bindings protein composition includes forexample a tag, for example a tag having an amino acid sequence of SEQ IDNO:15.

As used herein, the term “polypeptide” encompasses amino acid chains ofany length, including full length proteins (i.e., disease agents), inwhich the amino acid residues are linked by covalent peptide bonds. Apolypeptide comprises a portion of the binding agent, the entire bindingagent, or contains additional sequences. The polypeptides of the bindingagents of the present invention referred to herein as “isolated” arepolypeptides that are separated away and purified from other proteinsand cellular material of their source of origin. The compositions andmethods of the present invention also encompass variants of the abovepolypeptides and DNA molecules. A polypeptide “variant,” as used herein,is a polypeptide that differs from the recited polypeptide by having oneor more conservative substitutions and/or modifications, such that thefunctional ability of the binding agent to bind to the disease agenttarget is retained.

The present invention also encompasses proteins and polypeptides,variants thereof, or those having amino acid sequences analogous to theamino acid sequences of binding agents described herein. Suchpolypeptides are defined herein as analogs (e.g., homologues), ormutants or derivatives or variants. “Analogous” or “homologous” aminoacid sequences refer to amino acid sequences with sufficient identity ofany one of the amino acid sequences of the present invention so as topossess the biological activity (e.g., the ability to bind to thedisease agent target). For example, an analog polypeptide can beproduced with “silent” changes in the amino acid sequence wherein one,or more, amino acid residues differ from the amino acid residues of anyone of the sequence, yet still possesses the function or biologicalactivity of the polypeptide. The binding protein includes for example anamino acid having at least about 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%or 95%) identity or similarity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,56-87, 95 or combination thereof. Percent “identity” refers to theamount of identical nucleotides or amino acids between two nucleotidesor amino acid sequences, respectfully. As used herein, “percentsimilarity” refers to the amount of similar amino acids between twoamino acid sequences, i.e., having conservative amino acid changescompared to the original sequences, or to the amount of similarnucleotides between two nucleotide sequences.

In some embodiments, the invention pertains to one or more (e.g. about1, or about 2, or about 3, or about 4, or about 5, or about 6, or about7, or about 8, or about 9, or about 10, or about 15, or about 20)mutations to any of the sequences disclosed herein (e.g. as a variant ora portion or a homolog of such sequences). In various embodiments, avariant or portion or homolog has at least 30, 35, 40, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity toany of the sequences disclosed herein (e.g. a variant of SEQ ID NO: 163may include a sequence having at least 30, 35, 40, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to SEQ IDNO: 163). In various embodiments, one or more amino acid of any of thesequences disclosed herein is substituted with a naturally occurringamino acid, such as a hydrophilic amino acid (e.g. a polar andpositively charged hydrophilic amino acid, such as arginine (R) orlysine (K); a polar and neutral of charge hydrophilic amino acid, suchas asparagine (N), glutamine (Q), serine (S), threonine (T), proline(P), and cysteine (C), a polar and negatively charged hydrophilic aminoacid, such as aspartate (D) or glutamate (E), or an aromatic, polar andpositively charged hydrophilic amino acid, such as histidine (H)) or ahydrophobic amino acid (e.g. a hydrophobic, aliphatic amino acid such asglycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M),or valine (V), a hydrophobic, aromatic amino acid, such as phenylalanine(F), tryptophan (W), or tyrosine (Y) or a non-classical amino acid (e.g.selenocysteine, pyrrolysine, N-formylmethionine β-alanine, GABA andδ-Aminolevulinic acid. 4-Aminobenzoic acid (PABA), D-isomers of thecommon amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β methyl amino acids, C α-methyl aminoacids, N α-methyl amino acids, and amino acid analogs in general).

Referring to FIGS. 4 and 5, by comparing the B5 (SEQ ID NO: 24)polypeptide sequence to the other polypeptide sequences in the chart,the polypeptide sequence similarity is determined as follows: E-9 (SEQID NO: 38) is 74% similar, C5 (SEQ ID NO: 42) is 67% similar, B2 (SEQ IDNO: 40) is 68% similar, and F9 (SEQ ID NO: 44) is 73% similar. The BLASTwas done using default parameters on the NCBI website. Since these VHHshave been shown to compete with B5, i.e., for binding to the target, thepresent invention includes those sequences having a sequence similarityof at least about 65%. In like manner, by comparing the B5 (SEQ ID NO:23) nucleic acid sequence to the other nucleic acid sequences in thechart, the polypeptide sequence similarity is determined as follows: E-9(SEQ ID NO: 37) is 81% identical, C5 (SEQ ID NO: 41) is 75% identical,B2 (SEQ ID NO: 39) is 86% identical, and F9 (SEQ ID NO: 43) is 80%identical. The present invention includes those nucleic acid sequenceshaving a sequence identity of at least about 75%.

Homologous polypeptides are determined using methods known to those ofskill in the art. Initial homology searches are performed at NCBI bycomparison to sequences found in the GenBank, EMBL and SwissProtdatabases using, for example, the BLAST network service. Altschuler, S.F., et al., J. Mol. Biol., 215:403 (1990), Altschuler, S. F., NucleicAcids Res., 25:3389-3402 (1998). Computer analysis of nucleotidesequences can be performed using the MOTIFS and the FindPatternssubroutines of the Genetics Computing Group (GCG, version 8.0) software.Protein and/or nucleotide comparisons were performed according toHiggins and Sharp (Higgins, D. G. and Sharp, P. M., Gene, 199873:237-244, e.g., using default parameters). In certain embodiments, therecombinant multimeric binding protein acid sequence is an amino acidsequence that is substantially identical to sequences described herein,for example any of SEQ ID NOs: 56-87 and 95. The term “substantiallyidentical” is used herein to refer to a first amino acid sequence thatcontains a sufficient or minimum number of amino acid residues that areidentical to aligned amino acid residues in a second amino acid sequencesuch that the first and second amino acid sequences can have a commonstructural domain and/or common functional activity. For example, aminoacid sequences that contain a common structural domain having at leastabout 60% identity, or at least 75%, 85%, 95%, 96%, 98%, or 99%identity.

Calculations of sequence identity between sequences are performed asfollows. To determine the percent identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acidsequence for optimal alignment). The amino acid residues atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the proteins are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences are accomplished using a mathematical algorithm.Percent identity between two amino acid sequences is determined using analignment software program using the default parameters. Suitableprograms include, for example, CLUSTAL W by Thompson et al., Nuc. AcidsResearch 22:4673, 1994, BL2SEQ by Tatusova and Madden, FEMS Microbiol.Lett. 174:247, 1999, SAGA by Notredame and Higgins, Nuc. Acids Research24:1515, 1996, and DIALIGN by Morgenstern et al., Bioinformatics 14:290,1998.

The methods, compositions and kits described herein in variousembodiments include nucleotide sequence or an isolated nucleic acidmolecule (encoding the binding protein) having a nucleotide sequence ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 or combinations thereof. See FIGS. 1, 3and 4. As used herein, the terms “DNA molecule” or “nucleic acidmolecule” include both sense and anti-sense strands, cDNA, genomic DNA,recombinant DNA, RNA, and wholly or partially synthesized nucleic acidmolecules. A nucleotide “variant” is a sequence that differs from therecited nucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications are readily introducedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis as taught, for example, by Adelman et al. (DNA2:183, 1983). Nucleotide variants are naturally occurring allelicvariants, or non-naturally occurring variants. Variant nucleotidesequences in various embodiments exhibit at least about 70%, morepreferably at least about 80% and most preferably at least about 90%homology to the recited sequence. Such variant nucleotide sequenceshybridize to the recited nucleotide sequence under stringent conditions.In one embodiment, “stringent conditions” refers to prewashing in asolution of 6×SSC, 0.2% SDS; hybridizing at 65° Celsius, 6×SSC, 0.2% SDSovernight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDSat 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65°C.

The present invention also encompasses isolated nucleic acid sequencesthat encode the binding agents and in particular, those which encode apolypeptide molecule having an amino acid sequence of SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 56-87, 95 or combinations thereof.

As used herein, an “isolated” nucleotide sequence is a sequence that isnot flanked by nucleotide sequences which in nature flank the gene ornucleotide sequence (e.g., as in genomic sequences) and/or has beencompletely or partially purified from other transcribed sequences (e.g.,as in a cDNA or RNA library). Thus, an isolated gene or nucleotidesequence can include a gene or nucleotide sequence which is synthesizedchemically or by recombinant means. Nucleic acid constructs contained ina vector are included in the definition of “isolated” as used herein.Also, isolated nucleotide sequences include recombinant nucleic acidmolecules and heterologous host cells, as well as partially orsubstantially or purified nucleic acid molecules in solution. Thenucleic acid sequences of the binding agents of the present inventioninclude homologous nucleic acid sequences. “Analogous” or “homologous”nucleic acid sequences refer to nucleic acid sequences with sufficientidentity of any one of the nucleic acid sequences described herein, suchthat once encoded into polypeptides, they possess the biologicalactivity of any one of the binding agents described herein. Inparticular, the present invention is directed to nucleic acid moleculeshaving at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identitywith SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51 or combinations thereof.

Also encompassed by the present invention are nucleic acid sequences,DNA or RNA, which are substantially complementary to the DNA sequencesencoding the polypeptides of the present invention, and whichspecifically hybridize with their DNA sequences under conditions ofstringency known to those of skill in the art. As defined herein,substantially complementary means that the nucleotide sequence of thenucleic acid need not reflect the exact sequence of the encodingoriginal sequences, but must be sufficiently similar in sequence topermit hybridization with nucleic acid sequence under high stringencyconditions. For example, non-complementary bases can be interspersed ina nucleotide sequence, or the sequences can be longer or shorter thanthe nucleic acid sequence, provided that the sequence has a sufficientnumber of bases complementary to the sequence to allow hybridizationtherewith. Conditions for stringency are described in e.g., Ausubel, F.M., et al., Current Protocols in Molecular Biology, (Current Protocol,1994), and Brown, et al., Nature, 366:575 (1993); and further defined inconjunction with certain assays.

The invention also provides vectors, plasmids or viruses containing oneor more of the nucleic acid molecules having the sequence of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51 or combinations thereof). Suitable vectors foruse in eukaryotic and prokaryotic cells are known in the art and arecommercially available or readily prepared by a skilled artisan.Additional vectors can also be found, for example, in Ausubel, F. M., etal., Current Protocols in Molecular Biology, (Current Protocol, 1994)and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED.(1989).

Any of a variety of expression vectors known to those of ordinary skillin the art can be employed to express recombinant polypeptides of thisinvention. Expression can be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast and higher eukaryotic cells.Preferably, the host cells employed are E. coli, yeast, insect cells, ora mammalian cell line such as COS or CHO. The DNA sequences expressed inthis manner can encode any of the polypeptides described hereinincluding variants thereof.

Uses of plasmids, vectors or viruses containing the nucleic acids of thepresent invention include generation of mRNA or protein in vitro or invivo. In related embodiments, the methods, compositions and kitsencompass host cells transformed with the plasmids, vectors or virusesdescribed above. Nucleic acid molecules can be inserted into a constructwhich can, optionally, replicate and/or integrate into a recombinanthost cell, by known methods. The host cell can be a eukaryote orprokaryote and includes, for example, yeast (such as Pichia pastoris orSaccharomyces cerevisiae), bacteria (such as E. coli, or Bacillussubtilis), animal cells or tissue, insect 519 cells (such asbaculoviruses infected SF9 cells) or mammalian cells (somatic orembryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamsterovary cells, HeLa cells, human 293 cells and monkey COS-7 cells). Hostcells suitable in the present invention also include a mammalian cell, abacterial cell, a yeast cell, an insect cell, and a plant cell.

The nucleic acid molecule can be incorporated or inserted into the hostcell by known methods. Examples of suitable methods of transfecting ortransforming cells include calcium phosphate precipitation,electroporation, microinjection, infection, lipofection and directuptake. “Transformation” or “transfection” as used herein refers to theacquisition of new or altered genetic features by incorporation ofadditional nucleic acids, e.g., DNA. “Expression” of the geneticinformation of a host cell is a term of art which refers to the directedtranscription of DNA to generate RNA which is translated into apolypeptide. Methods for preparing such recombinant host cells andincorporating nucleic acids are described in more detail in Sambrook etal., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) andAusubel, et al. “Current Protocols in Molecular Biology,” (1992), forexample.

The host cell is maintained under suitable conditions for expression andrecovery of the polypeptides of the present invention. In certainembodiments, the cells are maintained in a suitable buffer and/or growthmedium or nutrient source for growth of the cells and expression of thegene product(s). The growth media are not critical to the invention, aregenerally known in the art and include sources of carbon, nitrogen andsulfur. Examples include Luria-Bertani broth, Superbroth, Dulbecco'sModified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media.The growth media can contain a buffer, the selection of which is notcritical to the invention. The pH of the buffered Media can be selectedand is generally one tolerated by or optimal for growth for the hostcell.

The host cell is maintained under a suitable temperature and atmosphere.Alternatively, the host cell is aerobic and the host cell is maintainedunder atmospheric conditions or other suitable conditions for growth.The temperature is selected so that the host cell tolerates the processand is for example, between about 13-40° Celsius.

The invention having now been fully described, it is further illustratedby the following claims and by the examples, which are found, in part,in a paper published in the Public Library of Science (PLoS) One andentitled, “A Novel Strategy for Development of Recombinant AntitoxinTherapeutics Tested in a Mouse Botulism Model”, co-authored by JeanMukherjee, Jacqueline M. Tremblay, Clinton E. Leysath, Kwasi Ofori,Karen Baldwin, Xiaochuan Feng, Daniela Bedenice, Robert P. Webb, PatrickM. Wright, Leonard A. Smith, Saul Tzipori, and Charles B. Shoemaker (12pages; Mukherjee J et al. 2012 PLoS ONE 7(1): e29941.doi:10.1371/journal.pone.0029941). This published paper is herebyincorporated by reference herein in its entirety.

Plants species have evolved chemical defenses against other organisms(Linskens, Hans F.; Jackson, John F. (Eds.) Plant Toxin Analysis 1992,XXVI, 389 p. 33 illus). Plants contain and secrete a variety of toxiccompounds sometimes referred to as “secondary compounds” that affect thebehavior and productivity of wild and domestic animals. Classes of toxiccompounds include soluble phenolics, alkaloids, and terpenoids. Solublephenolics include flavonoids, isoflavonoids, and hydrolysable andcondensed tannins.

Exemplary plants toxin molecules that in certain embodiments are treatedusing the compositions, methods and kits described herein are: Akar saga(Abrus precatorius), Deathcamas, Amianthium Angel's Trumpet(Brugmansia), Angel Wings (Caladium), Anticlea, Autumn crocus (Colchicumautumnale), Azalea (Rhododendron), Bittersweet nightshade (Solanumdulcamara), Black hellebore (Helleborus niger), Black locust (Robiniapseudoacacia), Black nightshade (Solanum nigrum), Bleeding heart(Dicentra cucullaria), Blind-your-eye mangrove (Excoecaria agallocha),Blister Bush (Peucedanum galbanum), Bloodroot (Sanguinaria canadensis),Blue-green algae (Cyanobacteria), Bobbins (Arum maculatum), Bracken(Pteridium aquilinum), Broom (Cytisus scoparius), calabar bean(Physostigma venenosum), castor bean, Christmas rose (Helleborus niger),Columbine (Aquilegia), Corn cockle (Agrostemma githago), corn lily(veratrum), cowbane (Cicuta), cows and bulls (Arum maculatum), crab'seye (Abrus precatorius), cuckoo-pint (Arum maculatum), daffodil(Narcissus), Darnel (Lolium temulentum), Deadly nightshade (Atropabelladonna), Devils and angels (Arum maculatum), False acacia (Robiniapseudoacacia), False hellebore (Veratrum), Foxglove (Digitalispurpurea), Frangipani (Plumeria), Doll's eyes (Actaea pachypoda),Dumbcane (Dieffenbachia), Dutchman's breeches (Dicentra cucullaria),Elder/Elderberry (Sambucus), Giant hogweed (Heracleum mantegazzianum),Giddee giddee, Gifblaar (Dichapetalum cymosum), Greater celandine(Chelidonium majus), Gympie gympie (Dendrocnide moroides), Heart ofJesus (Caladium), hemlock (Conium maculatum), hemlock water-dropwort(Oenanthe crocata), henbane (Hyoscyamus niger), Horse chestnut (Aesculushippocastanum), Holly (Ilex aquifolium), Hyacinth (Hyacinthusorientalis), Indian licorice, Jack in the pulpit, Jamestown weed,jequirity, Jerusalem cherry, Jimson weed, John Crow bead, Jumbie bead,Lily of the Valley, Lords and Ladies, Madiera winter cherry, Mayapple,Meadow saffron, Milky mangrove, Monkshood, Moonseed, Passion flower,Plumeria, Poison hemlock, Poison ivy, Poison oak, Poison parsnip, Poisonsumac, Poison ryegrass, Pokeweed, Precatory bean, Privet, ragwort,redoul, River poison tree, Robinia pseudoacacia (also known as blacklocust and false acacia), Rosary pea, Sosnowsky's Hogweed, Spindle tree,Starch-root, Stenanthium, Stinging tree, Stinkweed, Strychnine tree,Suicide tree (Cerbera odollam), thorn apple, Toxicoscordion, Wake robin,Water hemlock, White baneberry, White snakeroot, Wild arum, Wintercherry, Wolfsbane, Yellow Jessamine, Yew, and Zigadenus

Abrus precatorius is known commonly as jequirity, crab's eye, rosarypea, ‘John Crow’ bead, precatory bean, Indian licorice, akar saga,giddee giddee, jumbie bead, ruti, and weather plant. The attractiveseeds (usually about the size of a ladybug, glossy red with one blackdot) contain abrin, which is related to ricin, a very potent toxicsubstance to humans as a single seed can kill an adult human. Symptomsof poisoning include nausea, vomiting, convulsions, liver failure, anddeath, usually after several days. The seeds have been used as beads injewelry, which is dangerous; inhaled dust is toxic and pinpricks can befatal. The seeds are unfortunately attractive to children.

Aconitum species, commonly called aconite, wolfsbane and monkshood arepoisonous even by casual skin contact should be avoided; symptomsinclude numbness, tingling, and cardiac irregularity. The toxin is analkaloid called aconitine, which disables nerves, lowers blood pressure,and can stop the heart. It has been used as poison for bullets (byGermany in WWII), as a bait and arrow poison (ancient Greece), and topoison water supplies (reports from ancient Asia). If ingested, itusually causes burning, tingling, and numbness in the mouth, followed byvomiting and nervous excitement.

Actaea pachypoda known as doll's eyes or white baneberry are poisonousberries, and other parts of the plant are toxic. Consumption of theberries has a sedative effect on cardiac muscle tissue and can causecardiac arrest.

Adam and Eve (Arum maculatum) is a common woodland plant species of theAraceae family. It is widespread across temperate northern Europe and isknown by an abundance of common names including Wild arum, Lords andLadies, Devils and Angels, Cows and Bulls, Cuckoo-Pint, Adam and Eve,Bobbins, Naked Boys, Starch-Root and Wake Robin, Adenium obesum (alsoknown as sabi star, kudu or desert-rose). The plant exudes a highlytoxic sap which is used by the Meridian High and Hadza in Tanzania tocoat arrow-tips for hunting.

Aesculus hippocastanum (horse-chestnut) produces a toxin causing nausea,muscle twitches, and sometimes paralysis. Ageratina altissima (whitesnakeroot) produces a toxin, causing nausea and vomiting. Milk fromcattle that have eaten white snakeroot can sicken or kill humans.Aquilegia (columbine) seeds and roots that contain cardiogenic toxinscausing both severe gastroenteritis and heart palpitations if consumed,and poisoning by this plant is often fatal. Areca catechu (betel nutpalm and pinyang) a nut contains an alkaloid related to nicotine whichis addictive, mildly intoxicating, and if swallowed causes nausea. Useis correlated with mouth cancer, asthma and heart disease. Arummaculatum (jack in the pulpit) bright red berries contain oxalates ofsaponins and causes skin, mouth and throat irritation, resulting inswelling, burning pain, breathing difficulties and stomach upset. Atropabelladonna (deadly nightshade, and belladonna) is one of the most toxicplants found in the Western hemisphere, producing tropane alkaloidsincluding atropine, hyoscine (scopolamine), and hyoscyamine, which haveanticholinergic properties. The consumption of two to five berries bychildren and ten to twenty berries by adults can be lethal. The symptomsof poisoning include dilated pupils, sensitivity to light, blurredvision, tachycardia, loss of balance, staggering, headache, rash,flushing, dry mouth and throat, slurred speech, urinary retention,constipation, confusion, hallucinations, delirium, and convulsions.Ingestion of a single leaf of the plant can be fatal to an adult, andcasual contact with the leaves causes skin pustules. The berries posethe greatest danger to children because they look attractive and have asomewhat sweet taste. In 2009 a case of A. belladonna mistaken forblueberries, with six berries ingested by an adult woman, resulted insevere anticholinergic syndrome. A. belladonna is toxic also to manydomestic animals, causing narcosis and paralysis. Brugmansia (angel'strumpet) contains the tropane alkaloids scopolamine and atropine, andcan be fatal.

Caladium (commonly known as angel wings, elephant ear and heart ofJesus) produces symptoms such as generally irritation, pain, andswelling of tissues in subjects. If the mouth or tongue swell, breathingmay be fatally blocked. Cerbera odollam (suicide tree) produces seedsthat contain cerberin, a potent alkaloid toxin related to digoxin. Thepoison blocks the calcium ion channels in heart muscle, causingdisruption of the heart beat which is typically fatal. Chelidonium majusalso known as greater celandine is toxic in moderate doses as itcontains a range of isoquinoline alkaloids. The main alkaloid present inthe herb and root is coptisine, with berberine, chelidonine,sanguinarine and chelerythrine also present. Sanguinarine isparticularly toxic with an LD₅₀ of only 18 mg per kg body weight. Cicuta(water hemlock, cowbane, wild carrot, snakeweed, poison parsnip, falseparsley, children's bane and death-of-man) is extremely poisonous andcontains the toxin cicutoxin, a central nervous system stimulant,resulting in seizures. Colchicum autumnale (autumn crocus and meadowsaffron) bulbs contain colchicine, having poisoning symptoms thatinclude burning in the mouth and throat, fever, vomiting, diarrhea,abdominal pain and kidney failure. There is no specific antidote forcolchicine, although various treatments do exist. Conium maculatum(commonly known as hemlock, poison hemlock, spotted parsley, spottedcowbane, bad-man's oatmeal, poison snakeweed and beaver poison) containsthe alkaloid coniine which causes stomach pains, vomiting, andprogressive paralysis of the central nervous system. Consolida commonlyknown as larkspur is a poisonous plant that causes nausea, muscletwitches, paralysis and is often fatal.

Convallaria majalis (lily of the valley) is a poisonous woodlandflowering plant that contains cardiac glycosides fatal in humans.Coriaria myrtifolia (redoul) contains the toxin coriamyrtin. Ingestionof this plant produces digestive, neurological and respiratory problems.The poisonous fruit resemble blackberries and are often mistakenly eatenby children and adults. Cyanobacteria, a phylum of bacteria, is commonlyknown as blue-green algae. Many different species, including Anacystiscynea and Anabaena circinalis, produce several different toxins knowncollectively as cyanotoxins. Cyanotoxins include neurotoxins,hepatotoxins, endotoxins and cytotoxins. Cytisus scoparius (commonlyknown as broom or common broom) contains toxic alkaloids that depressthe heart and nervous system. The alkaloid sparteine is a class 1aantiarrhythmic agent and a sodium channel blocker. The berries of Daphneare either red or yellow and are poisonous, causing burns to mouth anddigestive tract, followed by coma. Datura contains the alkaloidsscopolamine and atropine. Datura has been used as a hallucinogenic drugby the native peoples of the Americas and others. Incorrect consumptionof this plant can lead to death. Datura stramonium (jimson weed, thornapple, stinkweed and Jamestown weed) causes abnormal thirst, visiondistortions, delirium, incoherence, and coma.

Deathcamas, including Amianthium, Anticlea, Stenanthium, Toxicoscordionand Zigadenus, are poisonous in many cases due to the presence ofalkaloids in the plants. Ingestion of the plant by grazing animals, suchas sheep and cattle, often results in death.

Delphinium (also known as larkspur) contains the alkaloid delsoline.Young plants and seeds of Delphinium are poisonous, causing nausea,muscle twitches, and paralysis. Dendrocnide moroides (also known asstinging tree and gympie gympie) causes a painful sting when touched andin some cases of widespread contact may be fatal. The stinging may lastfor several days and is exacerbated by touching, rubbing, and cold.Dicentra cucullaria (also known as bleeding heart and Dutchman'sbreeches) has leaves and roots that are poisonous and cause convulsionsand other nervous symptoms. Dichapetalum cymosum (also known asgifblaar) is a well known as a livestock poison in South Africa. Theplant contains the metabolic poison fluoroacetic acid. Dieffenbachia (ahouseplant dumbcane) causes intense burning, irritation, and immobilityof the tongue, mouth, and throat. Swelling can be severe enough to blockbreathing, leading to death. Digitalis purpurea (foxglove) leaves,seeds, and flowers are poisonous, containing cardiac or other steroidglycosides. These cause irregular heartbeat, general digestive upset,and confusion. Euonymus europaeus (commonly known as spindle, Europeanspindle or spindle tree). produces a poisonous fruit that containsamongst other substances, the alkaloids theobromine and caffeine, aswell as an extremely bitter terpene. Poisoning by this plant is morecommon in young children, who are enticed by the brightly-coloured fruitof the plant. Ingestion of the fruit results in liver and kidney damageand even death.

Excoecaria agallocha (milky mangrove) has a milky sap that causes skinirritation and blistering. Eye contact with the sap can even causetemporary blindness. Gelsemium sempervirens commonly known as yellowjessamine is poisonous, causing nausea, vomiting and even death. Hederahelix (English ivy) contains leaves and berries that can be poisonous,causing stomach pains, labored breathing, possible coma. Helleborusniger (Christmas rose) contains protoanemonin or ranunculin, which hasan acrid taste and can cause burning of the eyes, mouth and throat, oralulceration, gastroenteritis and hematemesis. Heracleum mantegazzianum(giant hogweed) produces a sap that is phototoxic, causingphytophotodermatitis (severe skin inflammations) when affected skin isexposed to sunlight or to UV-rays. Presence of minute amounts of sap inthe eyes can lead to temporary or even permanent blindness. Hippomanemancinella (manchineel) contains toxic phorbol esters typical of theEuphorbiaceae plant family. Contact with the milky white sap of theplant produces strong allergic dermatitis. The fruit is fatal if eaten.Hyacinthus orientalis (hyacinth) bulbs are poisonous, causing nausea,vomiting, gasping, convulsions, and possibly death. Even handling thebulbs can cause skin irritation.

Hyoscyamus niger (henbane) has seeds and foliage contain hyoscyamine,scopolamine and other tropane alkaloids that produces dilated pupils,hallucinations, increased heart rate, convulsions, vomiting,hypertension and ataxia. Ilex aquifolium (European holly) berries causegastroenteritis, resulting in nausea, vomiting and diarrhea. Jacobaeavulgaris (ragwort) contains alkaloids, including jacobine, jaconine,jacozine, otosenine, retrorsine, seneciphylline, senecionine, andsenkirkine. Kalanchoe delagoensis (mother of millions) containsbufadienolide cardiac glycosides which cause cardiac poisoning,particularly in grazing animals. Kalmia latifolia (mountain laurel)contains andromedotoxin and arbutin. The green parts of the plant,flowers, twigs, and pollen are all toxic, and symptoms of toxicity beginto appear about six hours following ingestion. Poisoning producesanorexia, repeated swallowing, profuse salivation, depression,uncoordination, vomiting, frequent defecation, watering of the eyes,irregular or difficulty breathing, weakness, cardiac distress,convulsions, coma, and eventually death. Laburnum produces seeds thatare poisonous and are lethal if consumed in excess. The main toxin inthe seeds is cytisine, a nicotinic receptor agonist. Symptoms ofpoisoning may include intense sleepiness, vomiting, excitement,staggering, convulsive movements, slight frothing at the mouth,unequally dilated pupils, coma and death. Ligustrum (privet) berries andleaves that are poisonous. The berries contain syringin, which causesdigestive disturbances, nervous symptoms. Privet is one of severalplants which are poisonous to horses. Lolium temulentum (poisonryegrass) produces seeds that contain the alkaloids temuline andloliine. The fungus ergot grow on the seed heads of rye grasses, as anadditional source of toxicity.

Mango peel and sap contains urushiol, the chemical in poison ivy andpoison sumac that can cause urushiol-induced contact dermatitis insusceptible people. Cross-reactions between mango contact allergens andurushiol have been observed. Those with a history of poison ivy orpoison oak contact dermatitis may be most at risk for such an allergicreaction. Narcissus bulbs and stems are poisonous, and cause nausea,vomiting, diarrhea, headaches, vomiting, and blurred vision.

Oenanthe crocata (hemlock water dropwort) contains oenanthotoxin in thestems and especially the carbohydrate-rich roots that are poisonous.Peucedanum galbanum (commonly known as blister bush) is poisonous andcontact to the body causes painful blistering that is intensified withexposure to sunlight. Physostigma venenosum (calabar bean) containsparasympathomimetic alkaloid physostigmine toxin, a reversiblecholinesterase inhibitor. Symptoms of poisoning include copious saliva,nausea, vomiting, diarrhea, anorexia, dizziness, headache, stomach pain,sweating, dyspepsia and seizures. Phytolacca (pokeweed) leaves, berriesand roots contain phytolaccatoxin and phytolaccigenin. Ingestion ofpoisonous parts of the plant cause severe stomach cramping, persistentdiarrhoea, nausea, vomiting (sometimes bloody vomiting), slow anddifficult breathing, weakness, spasms, hypertension, severe convulsions,and even death. Podophyllum peltatum (mayapple) contains thenon-alkaloid toxin podophyllotoxin, which causes diarrhea, severedigestive upset. Pteridium aquilinum (commonly known as bracken) ifingested is carcinogenic to humans and animals such as mice, rats,horses and cattle. The carcinogenic compound in the pant is ptaquiloside(PTQ), which can leach from the plant into the water supply. Pteridiumaquilinum (African sumac) is closely related to poison ivy. The treecontains low levels of a highly irritating oil with urushiol. Skinreactions to contacting the plan include blisters and rashes that befurther spread by contacting clothing of an exposed subjects. The smokeof burning Rhus lancia can cause reactions in the lungs, and can befatal. Ricinus communis (castor oil plant) seeds contain ricin, anextremely toxic water-soluble protein, ricinine, an alkaloid, and anirritant oil.

Sambucus (commonly known as elder or elderberry) roots are poisonous andcause nausea and digestive upset. Sanguinaria canadensis (bloodroot)rhizome or stem contains morphine-like benzylisoquinoline alkaloids, andthe toxin sanguinarine. Sanguinarine kills animal cells by blocking theaction of Na+/K+-ATPase transmembrane proteins. Solanum dulcamara(bittersweet nightshade) contains solanine which causes fatigue,paralysis, convulsions, and diarrhea in subjects exposed to the plant.Solanum nigrum (black nightshade) contains the toxic glycoalkaloidsolanine. Solanine poisoning is primarily displayed by gastrointestinaland neurological disorders. Symptoms include nausea, diarrhea, vomiting,stomach cramps, burning of the throat, cardiac dysrhythmia, headache anddizziness. Taxus baccata (yew) contains toxic taxanes. The plant seedsthemselves are particularly toxic if chewed. Toxicodendron contain ahighly irritating oil with urushiol. Species of toxicodendrons includeToxicodendron radicans (commonly known as poison ivy), Toxicodendrondiversilobum (commonly known as poison-oak), and Toxicodendron vernix(commonly known as poison sumac. These plants cause skin reactions suchas blisters and rashes. Urtica ferox (ongaonga) cause a painful stingthat lasts several days. Veratrum (false hellebore and corn lily)contain a highly toxic steroidal alkaloids (e.g. veratridine) thatactivate sodium ion channels and cause rapid cardiac failure and deathif ingested. Symptoms typically occur between 30 minutes and four hoursafter ingestion and include nausea and vomiting, abdominal pain,numbness, headache, sweating, muscle weakness, bradycardia, hypotension,cardiac arrhythmia, and seizures. Xanthium (commonly known as cocklebur)includes X. strumarium, a native of North America, that is poisonous tolivestock, including horses, cattle, and sheep. The seedlings and seedsare the most toxic parts of the plants and produce unsteadiness andweakness, depression, nausea and vomiting, twisting of the neck muscles,rapid and weak pulse, difficulty breathing, and eventually death.Zantedeschia (Calla lily) contain calcium oxalate and other toxins thatproducing irritation and swelling of the mouth and throat, acutevomiting and diarrhea.

Endosperm of castor seeds contain two highly toxic proteins (Ghetie etal. U.S. Pat. No. 5,578,706 issued Nov. 26, 1996; and Lord et al., 1994The FASEB Journal 8, 201-208, each of which is incorporated by referenceherein in its entirey). R. communis agglutinin (RCA), a 120 kDahemagglutinin lectin, and ricin, a 65 kDa cytotoxic lectin, are lethalto eukaryotic cells. Ricin has two polypeptide chains, A and B, whichtogether are highly lethal to mammalian cells. Agglutinin protein hasfour polypeptides, linked by disulfide bonds (Butterworth et al., 1983Eur. J. Biochem. 137, 57-65), two of which are similar to the A chain(ricin A chain; RTA) and two similar to the B chain of ricin (ricin Bchain, RTB). The A and B chains of ricin, together. Ricin E is a variantof the ricin toxin, having an A chain similar to ricin and a B chain,which is a hybrid of the ricin and RCA and B chains (Ladin et al. 1987Plant Molecular Biology 9: 287-295).

Ricin A chain (32 kDa) has a ribosome-inactivating activity (Lord etal., 1994 The FASEB Journal 8, 201-208), irreversibly altering theribosomal RNA subunits involved in translation. The A chain specificallybinds 28S ribosomal subunits; the A chain requires the B chain to enterthe cells as a heterodimeric toxin.

Ricin's B chain is a lectin which specifically binds glycoproteins andglycolipids on the cell surface terminating in galactose orN-acetylgalactosamine (Lord et al., 1994 The FASEB Journal 8, 201-208).The B chain binds more strongly to complex galatosides than to simplesugars. The B chain has four disulfide bonds and has agalactose/N-acetylgalactosamine binding activity. The N-terminal andC-terminal halves of the B chain contain 41 homologous pairs of aminoacids when the two disulfide bonds in each half are aligned, yielding abilobal structure with two galactose binding sites. Subdomains formed bythe four disulfide bonds represent a conserved peptide which is repeatedfour times (Roberts et al., 1985 Journal of Biological Chemistry 260,15682-8.). Up to 108 ricin B chains bind to an individual cell byhydrogen bonds (Lord et al., 1994 The FASEB Journal 8, 201-208). The Bchain attaches to the eukaryotic cell and the intact toxin enters thecell by receptor mediated endocytosis (Bilge et al., 1995 Journal ofBiological Chemistry 1995; 270(40):23720-23725). The B chain protectsthe A chain from proteolytic activities of lysosomes and cathepsins.Mannose residues attached to ricin are bound by cellular mannosereceptors and initiate endocytosis (Montfort et al., 1987 Journal ofBiological Chemistry 262, 5398-403).

A portion of the various embodiments herein are described in Tremblay etal. Figures in Tremblay et al. show that VHH domains neutralize Stx1and/or Stx2.

Tremblay et al. is a published paper that describes heavy-chain-onlycamelid antibodies (VHHs) that were found to neutralize Stx1 and/or Stx2in cell-based assays. It was observed that VHHs are effectiveheterodimer toxin neutralizing agents. The VHHs containing two linkedStx1-neutralizing VHHs or two Stx2-neutralizing VHHs were observed to bemuch more effective at neutralizing Stx in cell-based assays and in vivoin mouse subjects than a mixture of the two component monomers, e.g.,heterodimer A5/D10 was found to have greater neutralization ability thana mixture of VHH A5 and VHH D10.

Further, clearance of toxins was observed to have been promoted by thepresence of anti-tag monoclonal antibody co-administered with aVHH-based toxin neutralizing agent, referred to herein as the “effectorAb”. The effector Ab binds to a common epitopic tag which was engineeredto be located on each of the two VHH heterodimer molecules that bind tothe toxin. It was observed that co-administration of effector Ab (or aplurality of effector Abs) substantially improved the efficacy of Stxtoxin neutralizing agents, and prevention of death and kidney damage inmice following challenge with Stx1 or Stx2. A single toxin neutralizingagent composed of a double-tagged VHH heterotrimer co-administered witheffector Ab was observed to effectively prevent all symptoms ofintoxication due to Stx1 and Stx2. The VHH heterotrimer was engineeredto contain a Stx1-specific VHH, a Stx2-specific VHH, and a Stx1/Stx2cross-specific VHH. Without being limited by any particular theory ormechanism of action, it is envisioned that availability of simple,defined, recombinant proteins as described herein and in Tremblay et al.results in cost-effective protection against diseases such as hemolyticuremic syndrome, opening up new therapeutic approaches to managing thesediseases.

A portion of the various embodiments herein are described in David J.Vance, Jacqueline M. Tremblay, Nicholas J. Mantis, and Charles B.Shoemaker; “Stepwise Engineering of Heterodimeric Single Domain CamelidVHH Antibodies That Passively Protect Mice from Ricin Toxin”; Journal ofBiological Chemistry; 288(51):365-36547 (2013) (hereinafter Vance etal.) which is incorporated herein by reference in its entireties.

A skilled person will recognize that many suitable variations of themethods may be substituted for or used in addition to those describedabove and in the claims. It should be understood that the implementationof other variations and modifications of the embodiments of theinvention and its various aspects will be apparent to one skilled in theart, and that the invention is not limited by the specific embodimentsdescribed herein and in the claims. Therefore, it is contemplated tocover the present embodiments of the invention and any and allmodifications, variations, or equivalents that fall within the truespirit and scope of the basic underlying principles disclosed andclaimed herein.

The following examples and claims are illustrative and are not meant tobe further limiting. Those skilled in the art will recognize or be ableto ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references including issued patents and publishedpatent applications cited in this application are hereby incorporated byreference.

EXAMPLES Example 1: Toxins and Reagents

Botulinum neurotoxin serotype A1 (BoNT/A) and serotype B (BoNT/B) wereobtained from Metabiologics Inc. Each batch of toxin was calibrated toestablish the LD₅₀ dose in mice and stored in aliquots at −80° C. untiluse. Purified recombinant BoNT serotype A1 and B holotoxins containingmutations rendering them catalytically inactive (ciBoNTA, ciBoNTB)obtained. Sheep anti-BoNT/A1 antiserum was produced by immunization ofsheep with BoNT/A1 toxoid followed by BoNT/A1 holotoxin. Less than 1 μlof this sheep antitoxin serum protects mice from lethality whenco-administered with 10,000-fold the LD₅₀ of BoNT/A1. Reagents forWestern blotting were purchased from KPL (Gaithersburg, Md.).

C. difficile holotoxins TcdA and TcdB were generated by transformationof shuttle vectors pHis1522 (pHis-TcdA and pHis-TcdB respectively) intoB. megaterium described in Yang et al. 2008 BMC Microbiolgoy 8:192.Point mutations were introduced into conserved amino acids that areresponsible for binding to the substrate, uridine diphosphoglucose(UDP-Glucose), in order to generate GT-deficient holotoxins. To generateGT-mutant holotoxin A, a unique restriction enzyme (BamHI) site wasdesigned and constructed between sequences encoding GT and CPD domainsusing overlapping PCR. The primer sets used were:

pHis-F (5′-TTTGTTTATCCACCGAACTAAG-3′; SEQ ID NO: 90), Barn-R(5′-TCTTCAGAAAGGGATCCACCAG-3′; SEQ ID NO: 91), Barn-F(5′-TGGTGGATCCCTTTCTGAAGAC-3′; SEQ ID NO: 92), and Bpu-R(5′-ACTGCTCCAGTTTCCCAC-3′; SEQ ID NO: 93).

The final PCR product was digested with BsrGI and Bpu10I, and was usedto replace the corresponding sequence in pHis-TcdA. The resultingplasmid was designated pH-TxA-b. Sequences encoding triple mutations(W101A, D287N, and W519A) in the GT were synthesized by Geneart(Regensburg, Germany) and cloned into pH-TxA-b through BsrGI/BamHIdigestion. To generate the mutant holotoxin B construct, the sequencebetween BsrGI and NheI containing two point mutations (W102A and D288N)was synthesized and inserted into pHis-TcdB at the same restrictionenzyme sites, leading to a new plasmid pH-aTcdB. The mutant aTcdA andaTcdB were expressed and purified identical to the wild types in B.megaterium as described by Yang et al. 2008 BMC Microbiology 8:192. Thepurified aTcdA and aTcdB were used to immunize alpacas.

Example 2: Alpaca Immunization and VHH-Display Library Preparation

Purified, catalytically inactive mutant forms of full-length recombinantBoNT/A (ciBoNTA) and BoNT/B (ciBoNTB) proteins were obtained asdescribed in Webb et al. 2009 Vaccine 27: 4490-4497. Alpacas (twoanimals per immunization type) were immunized with either ciBoNTA orwith ciBoNTB. Additional alpacas were immunized with aTcdA or aTcdB. Theimmunization regimen employed 100 μg of protein in the primaryimmunization and 50 μg in three subsequent boosting immunizations atthree weekly intervals in aluminum hydroxide gel adjuvant in combinationwith oligodeoxynucleotides containing unmethylated CpG dinucleotides(alum/CpG; Superfos Biosector; Copenhagen, Denmark) adjuvant. Five daysfollowing the final boost immunization, blood from each animal wasobtained for lymphocyte preparation and VHH-display phage libraries wereprepared from the immunized alpacas as previously described (Maass etal. 2007 Int J Parasitol 37: 953-962 and Tremblay et al. 2010 Toxicon.56(6): 990-998). Independent clones (greater than 10⁶ total) wereprepared from B cells of alpacas successfully immunized with each of theBoNT immunogens.

Example 3: Anti-BoNT VHH Identification and Preparation

The VHH-display phage libraries were panned for binding to ciBoNTA orciBoNTB targets that were coated onto each well of a 12-well plate.Coating was performed by overnight incubation at 4° C. with one ml of a5 μg/ml target solution in PBS, followed by washing with PBS and twohours incubation at 37° C. with blocking agent (4% non-fat dried milkpowder in PBS). Panning, phage recovery and clone fingerprinting wereperformed as previously described (Ibid.). Based on phage ELISA signals,a total of 192 VHH clones were identified as strong candidate clones forbinding to BoNT/A, and 142 VHH clones were identified as strongpositives for binding to BoNT/B respectively. Of the strong positives,62 unique DNA fingerprints were identified among the VHHs selected forbinding to BoNT/A and 32 unique DNA fingerprints were identified forVHHs selected for binding to BoNT/B. DNA sequences of the VHH codingregions were obtained for each phage clone and compared for identifyinghomologies. Based on these data, twelve of the anti-BoNT/A VHHs andeleven anti-BoNT/B VHHs were identified as unlikely to have common Bcell clonal origins and were selected for protein expression. Expressionand purification of VHHs in E. coli as recombinant thioredoxin (Trx)fusion proteins containing hexahistidine was performed as previouslydescribed in Tremblay et al. 2010 Toxicon. 56(6): 990-998. Forheterodimers, DNA encoding two different VHHs were joined in framedownstream of Trx and separated by DNA encoding a fifteen amino acidflexible spacer having the amino acid sequence (GGGGS)₃. VHHs wereexpressed with a carboxyl terminal E-tag epitope. Furthermore, a numberof VHH expression constructions were engineered to contain a second copyof the E-tag by introducing the coding DNA in frame between the Trx andVHH domains. An example of a Trx fusion to a VHH heterodimer with twoE-tags is ciA-H7/ciA-B5(2E) shown in FIG. 13 C.

Example 4: VHH Target Binding Competition Analysis

Phage displaying individual VHHs were prepared and titered by phagedilution ELISA for recognition of ciBoNTA or ciBoNTB using HRP/anti-M13Ab for detection (Maass et al. 2007 Int J Parasitol 37: 953-962). Adilution was selected for each phage preparation that produced a signalnear the top of the linear range of the ELISA signal. The selected phagedilution (100 μl) for each VHH-displayed phage preparation were added to96 well plate that has been coated with ciBoNTA or ciBoNTB and thenpre-incubated for 30 minutes with 100 μl of a 10 μg/ml solutioncontaining a purified Trx/VHH fusion protein test agent or control inPBS. After an hour, the wells were washed and phage binding wasdetected. Test VHHs that reduced target binding of phage-displayed VHHsby less than two-fold compared to controls were considered to recognizedistinct epitopes. Positive controls were prepared in which the Trx/VHHcompetitor contained the same VHH as displayed on phage and typicallyreduced the ELISA signal detected by greater than 95%.

Example 5: Characterization of VHH Binding Properties

VHHs were tested for binding to native or atoxic mutant BoNT holotoxinsby standard ELISA using plates coated with 100 μl of 1 μg/ml protein.VHHs were also tested for recognition of BoNT subunits by ELISA usingplates coated with 5 μg/ml purified recombinant BoNT light chain or 1μg/ml BoNT heavy chain. See Tremblay et al. 2010 Toxicon. 56(6):990-998. VHHs were also characterized for recognition of subunits byWestern blotting on BoNT holotoxin following SDS-PAGE electrophoresisunder reducing conditions. VHHs were detected with HRP-anti-E-tag mAb(GE Healthcare) by standard procedures.

Example 6: Kinetic Analysis by Surface Plasmon Resonance

Assays to assess the kinetic parameters of the VHHs were performed usinga ProteOn XPR36 Protein Interaction Array System (Bio-Rad, Hercules,Calif.) after immobilization of ciBoNT/A by amine coupling chemistryusing the manufacturer recommended protocol. Briefly, after activationof a GLH chip surface with a mixture of 0.4 M ethyl(dimethylaminopropyl) carbodiimide (EDC) and 0.1 MN-hydroxysulfosuccinimide (sulfo-NHS) injected for 300 s at 30 μL/min,ciBoNT/A was immobilized by passing a 60 μg/mL solution of the proteinat pH 5 over the surface for 180 s at 25 μL/min. The surface wasdeactivated with a 30 μL/min injection of 1 M ethanolamine for 300 s. Aconcentration series for each VHH (between 2.5 nM and 1000 nM, optimizedfor each antibody fragment) was passed over the surface at 100 μL/minfor 60 s, then dissociation was recorded for 600 s or 1200 s. Thesurface was then regenerated with a 36 s injection of 10 mM glycine, pH2.0 at 50 μL/min. The running buffer used for these assays was 10 mMHepes, pH 7.4, 150 mM NaCl, 0.005% Tween-20. Data was evaluated withProteOn Manager software (version 2.1.2) using the Langmuir interactionmodel.

Example 7: BoNT Neutralization Assay Using Primary Neurons

Neuronal granule cells from the pooled cerebella of either 7-8 day oldSprague-Dawley rats or 5-7 day old CD-1 mice were harvested (Skaper etal 1979 Dev Neurosci 2: 233-237) and cultured in 24 well plates asdescribed by Eubanks et al 2010 ACS Med Chem Lett 1: 268-272. After atleast a week of culture the well volumes were adjusted to 0.5 mlcontaining various VHH dilutions or buffer controls followed immediatelyby addition of BoNT/A in 0.5 ml to a final 10 pM. After overnight at 37°C., cells were harvested and the extent of SNAP25 cleavage assessed byWestern blot as previously described (Eubanks, L. M. et al. 2007 Proc.Natl. Acad. Sci. USA 104: 2602-2607).

Example 8: Mouse Toxin Lethality Assay

Female CD1 mice (Charles River) about 15-17 g each were received fromthree to five days prior to use. On the day each assay was initiated,mice were weighed and placed into groups in an effort to minimizeinter-group weight variation. Appropriate dilutions of the VHH agentswere prepared in PBS. BoNT holotoxins were separately prepared in PBS atthe desired doses. Amounts (600 μl) of VHH agent and (600 μl) of thetoxin were combined and incubated at room temperature for 30-60 minutes.An amount (200 μl) of each mixture was administered by intravenousinjection at time point zero to groups of mice (five mice per group).Mice were monitored at least four times per day and assessed forsymptoms of toxin exposure and lethality/survival. Moribund mice wereeuthanized. Time to onset of symptoms and time to death were establishedfor each mouse.

Example 9: Mouse Toxin Lethality Assay with Agents AdministeredPost-Intoxication

Groups of mice were prepared as described in the description of themouse toxin lethality assay. Subjects were administered 10 LD₅₀ ofBoNT/A by intraperitoneal injection. At indicated timespost-intoxication, mice were administered 200 ul of material (e.g., VHHmonomer or VHH heterodimer) in PBS by intravenous injection. Mice weremonitored for symptoms of intoxication and death as described herein.

Example 10: Single-Chain Fvs (scFv) that Recognize and Bind BoNT/A

To improve therapies that involve multiple monoclonal antibodies (mAbs)by using small recombinant peptide, protein or polynucleotide agentsthat have the same binding specificity as the mAbs, each of therecombinant binding agents is produced containing the same epitopic tag.A single mAb that recognizes the epitopic tag is co-administered topatients with the binding agents. The different agents bind to the sametargets as the multiple mAbs and the anti-tag mAb binds to these agentsthrough the epitopic tag. This permits delivery of the same therapeuticeffect that is achieved with multiple mAb therapy, but requires only asingle mAb. If desired, mAbs of different isotypes, or polyclonalanti-tag antibodies, could be used therapeutically to deliver differentimmune effector activities.

A number of small recombinant protein agents were generated. They werecalled single-chain Fvs (scFvs) and were observed to recognize botulinumneurotoxin serotype A (BoNT/A). These scFvs are recombinant proteinsthat represent the antigen combining region of an immunoglobulin.Several anti-BoNT/A scFvs were produced and were purified. Each scFvcontains the amino acid sequence (GAPVPYPDPLEPR; SEQ ID NO: 15) near thecarboxyl terminus which is an epitopic tag referred to herein as“E-tag.” An scFvs (scFv#2) was shown to neutralize BoNT/A in acell-based toxin assay (IC50˜7 nM). A second scFv (scFv#7) had little orno neutralization activity in the assay, and was found to bind to BoNT/Awith high affinity (Kd˜1 nM).

The scFvs were tested for their ability to protect mice from thebotulinum toxin BoNT/A by intravenous administration of the agents andtoxin. The two scFvs were administered individually or together, andwere given to mouse subjects with and without anti-E-tag mAb byintravenous administration. Each subject was administered a dose of 10LD₅₀ of BoNT/A (i.e., an amount of BoNT/A ten-fold the LD₅₀), five miceper group. The results are shown in Table 1.

TABLE 1 scFv administration with and without anti-tag antibodyalleviates toxin morbidity Agents Administered (dose) SurvivalObservations none 0% Death in less than a day scFv#2 (20 μg) 0% Deathdelayed about a day scFv#7 (20 μg) 0% Death delayed less than a dayscFv#2 (20 μg) + anti-E-tag mAb (25 μg) 100% Symptoms severe scFv#7 (20μg) + anti-E-tag mAb (25 μg) 0% Death delayed several days scFv#2 (10μg) + scFv#7 (10 μg) + anti-E- 100% No symptoms tag mAb (25 μg)

The results shown in Table 1 clearly show that a BoNT/A neutralizingscFv (scFv#2) alone did not significantly protect mice from the toxin.Subjects survived (100%) following co-administration scFV#2 and mAb thatrecognizes an epitopic tag (E-tag) on the scFv. More importantly,co-administering two scFvs, each with E-tag, and anti-tag mAbdramatically improved the protective effect.

Subjects were administered 10 LD₅₀ and lower doses of the scFvs and theanti-E-tag mAb, and were analyzed for percent survival. Further, twoadditional non-neutralizing anti-BoNT/A scFvs (scFv#3 and scFv#21) weretested in combination with the neutralizing scFv#2. Whether theanti-E-tag mAb would function upon administration at a different siteand time than the toxin was also tested.

The results in Table 2 confirm those data herein and further show thatthe mAb specific for the epitopic tag does not have to be pre-mixed withthe scFv containing the epitopic tag to be effective. In fact, doseswere administered at different sites and times. Combinations of twoscFvs (each with E-tags) and the single anti-E-tag mAb, provided greaterprotection than with one scFv alone. This synergistic protective effectoccurred using different scFvs and was observed at significantly lowerdoses of the scFvs or mAb than used in the data observed in Table 1.

TABLE 2 Anti-E-tag mAbs administered separately protected subjects fromtoxin Agents Administered (dose) Survival Observations none 0% Death inabout a day scFv#2 (10 μg) 0% Death delayed about 2 days scFv#2 (10μg) + anti-E-tag mAb (10 μg) 100% Symptoms moderate (mAb administeredintraperitoneally) scFv#2 (10 μg) + anti-E-tag mAb (10 μg) 100% Symptomsmild scFv#2 (10 μg) + anti-E-tag mAb (2 μg) 100% Symptoms mild scFv#2 (2μg) + anti-E-tag mAb (2 μg) 100% Symptoms moderate scFv#2 (5 μg) +scFv#7 (3 μg) + 100% No symptoms anti-E-tag mAb (10 μg) scFv#2 (1 μg) +scFv#7 (1 μg) + 100% No symptoms anti-E-tag mAb (10 μg) scFv#2 (5 μg) +scFv#3 (4 μg) + 100% No symptoms anti-E-tag mAb (10 μg) scFv#2 (5 μg) +scFv#21 (3 μg) + 100% No symptoms anti-E-tag mAb (10 μg)

Examples herein tested whether combinations of three and four scFvs withanti-tag mAb protect subjects from an amount of BoNT/A 100-fold,1000-fold, or 10,000-fold the LD₅₀, i.e., 100 LD₅₀ BoNT/A, 1000 LD₅₀BoNT/A or 10,000 LD₅₀ BoNT/A.

The data shown in Table 3 demonstrate the excellent potency of a taggedbinding agent as an antitoxin. Specifically, completely protection ofsubjects from even mild symptoms of intoxication by 1,000 LD₅₀ wasobserved using combinations of three or four scFvs with anti-E-tag mAb.Subjects were protected from lethality from a 10,000 LD₅₀ dose with acombination of four scFvs, although moderate symptoms were observed. Theability to protect mice receiving up to 10,000 LD₅₀ of BoNT/A isequivalent to the highest level of protection reported with pools ofdifferent anti-BoNT/A mAbs (Nowakowski et al, Proc Natl Acad Sci USA,99:11346-50).

TABLE 3 Combinations of scFv protect from 100, 1000, and 10,000 foldLD₅₀ BoNT/A doses in presence of 10 μg of anti-E-tag mAb BoNT/AAdditional agents administered (dose) Survival Observations   100 LD₅₀None 0% Death in less than a day   100 LD₅₀ scFv#2 (2 μg) + scFv#3 (2μg) + scFv#21 (2 μg) 100% No symptoms  1,000 LD₅₀ None 0% Death in lessthan a day  1,000 LD₅₀ scFv#2 (2 μg) + scFv#3 (2 μg) + scFv#21 (2 μg)100% No symptoms  1,000 LD₅₀ scFv#2 (2 μg) + scFv#3 (2 μg) + scFv#7 (2μg) + 100% No symptoms scFv#21 (2 μg) 10,000 LD₅₀ None 0% Death in a fewhours 10,000 LD₅₀ scFv#2 (2 μg) + scFv#3 (2 μg) + scFv#21 (2 μg) 0%Death delayed one day 10,000 LD₅₀ scFv#2 (2 μg) + scFv#3 (2 μg) + scFv#7(2 μg) + 100% Moderate symptoms scFv#21 (2 μg)

The next example tested efficacy of a binding agent containing twocopies of the epitopic tag. The anti-BoNT/A binding agent, scFv#7, wasengineered to contain another copy of the E-tag peptide. The E-tagpeptide was present on the carboxyl terminus of each scFv. An alteredversion of scFv#7 (called scFv#7-2E) was engineered to be identical toscFv#7 and to have an additional copy of the E-tag peptide fused to theamino terminus.

TABLE 4 Protection from BoNT/A using scFvs having multiple tag sequencesin presence of 10 μg of anti-E-tag mAb BoNT/A LD₅₀ Additional agentsadministered (1 μg each) Survival Observations 100 None 0% Death in lessthan 6 hours 100 scFv#2 + scFv#3 + scFv#7 100% No symptoms 100 scFv#2 +scFv#3 + scFv#7-2E 100% No symptoms 1,000 None 0% Death in less than 2hours 1,000 scFv#2 + scFv#3 + scFv#7 0% Death delayed 2 days 1,000scFv#2 + scFv#3 + scFv#7-2E 100% No symptoms 10,000 None 0% Death inless than 2 hours 10,000 scFv#2 + scFv#3 + scFv#7 0% Death delayed lessthan a day 10,000 scFv#2 + scFv#3 + scFv#7-2E 20% Death delayed manydays 10,000 scFv#2 + scFv#3 + scFv #21 + scFv#7 0% Death delayed 2 days10,000 scFv#2 + scFv#3 + scFv #21 + scFv#7-2E 100% Moderate symptoms

The results in Table 4 demonstrate that the binding agent with twoepitope tags dramatically improved the in vivo antitoxin efficacy of thetagged binding agent. With a combination of three scFvs, includingscFvs#2, scFvs#3 and scFvs#7 or scFvs#7-2E, clearly the use ofscFvs#7-2E was substantially superior in protection of mice to the useof scFvs#7 with only one E-tag. The improvement by presence of twocopies of tag was particularly evident in the groups of mice challengedwith 1,000 LD₅₀. In these groups, the triple combination ofscFv#2+scFv#3+scFv#7 was insufficient to allow survival of the mice.When scFv#7 was replaced with scFv#7-2E, all the mice survived withoutsymptoms. Furthermore, use of a pool of scFv#2+scFv#3+scFv#7-2Epermitted the survival of one of five mice challenged with 10,000 LD₅₀and delayed the death of the other mice by several days. The equivalentpool with scFv#7 having only one E-tag only delayed death for one day inmice challenged with 10,000 LD₅₀. Finally, an identical combination offour scFvs (#2, #3, #21 and #7) in which the efficacy using scFv#7 wascompared with scFv#7-2E. Administering only one μg of each scFv, thepresence of scFv#7 did not result in survival of mice challenged with10,000 LD₅₀, and the same combination the scFv#7-2E was protective.These data show that mice were effectively protected from high doses oftoxin by administering a smaller number of high affinity binding agents,each containing two or more epitope tags together with an anti-tag mAb.

The method herein improves therapeutic agent flexibility, provideshighly stable binding agents with long shelf life, substantially reducesthe cost of production, and permits commercially feasible therapeuticapplications that involve multiple target agents. Furthermore, thestrategy herein will permit much more rapid development of newantitoxins. The binding agents are much more quickly developed tocommercialization than mAbs. The single anti-tag mAb needed forco-administration is the same for therapies requiring different taggedbinding agents and thus can be pre-selected for its commercial scale upproperties and stockpiled in advance of the development of the bindingagents.

An immediate application is in anti-toxin therapy, an area of highinterest because of bioterrorist threats. For example, it is now thoughtthat effective prevention of botulinum intoxication using toxinneutralizing mAbs will require administration of three different mAbseach targeting the same toxin. Since there are at least seven differentbotulinum toxins, this suggests that 21 different mAbs (or more) mayneed to be stockpiled for use in the event of a major botulism outbreakas might occur through bioterror. Monoclonal antibodies are veryexpensive to produce and have relatively short shelf lives. Methods andcompositions herein would make it possible to produce 21 differentrecombinant binding agents, each having longer shelf-life and lowerproduction costs, and then stockpile only a single mAb. It is possiblethat this approach could open up many other mAb therapeutic strategiesthat involve multiple binding targets, but which have not been pursuedbecause of prohibitive development and production costs and poor productshelf life. Methods and compositions herein permit the use of mAbs ofdifferent antibody isotypes to be used with the same binding agents toprovide greater therapeutic flexibility.

Example 11: BoNT/a VHHs Binding Agents

VHH binding agents were identified, produced and purified that werespecific to each of botulinum neurotoxin serotype A (BoNT/A) andserotype B (BoNT/B). The VHHs made herein included nine amino acids atthe amino coding end and which are associated with the forward PCRprimer sequence. See FIG. 3 A-C for the sequences. These sequencesderive from ‘framework 1’ and include minor variants of the originalcoding sequence. The most common amino acid sequence is QVQLVESGG (SEQID NO: 16) and which is the amino acid sequence used in assays shown inFIG. 3 A-C.

At the carboxyl coding end of the VHHs either amino sequence, AHHSEDPS(SEQ ID NO: 17), or the amino sequence, EPKTPKPQ (SEQ ID NO: 18) islocated, present in the VHHs sequence as shown in FIG. 3 A-C, and thesewere observed to be interchangeable without loss of function. Identicalclones were identified from alpacas that vary only in the hinge sequenceand retain virtually the same target binding function. See also D. R.Maass et al. 2007 Journal of Immunological Methods 324:13-25.

As a result of the altered splicing, the amino acid sequence that joinsthe VH domain to the CH2 domain in heavy chain IgGs is called the“hinge” region, and is unique to this class of camelid antibodies (SeeD. R. Maass et al. 2007 Journal of Immunological Methods 324:13-25 whichis incorporated by reference in its entirety). The two distinct hingesequence types found in camels and llamas are referred to as the “short”hinge and the “long” hinge respectively. SEQ ID NO: 17 is a short hingesequence derived from a camel, and SEQ ID NO: 18 is a long hingesequence derived from a llama.

During screening for VHH binding agents, different coding sequences areidentified that display significant homology among randomly identifiedclones. VHH sequences that are homologous are predicted to be relatedand thus to recognize the same epitope on the target to which they havebeen shown to bind. Examples herein experimentally demonstrate epitoperecognition by methods for binding competition. These findingsdemonstrate that significant variation is permitted in VHH amino acidsequences without loss of target binding. An example of the extent ofvariation permitted is shown in FIG. 4 A-B. Each VHH identified in FIG.4 A-B as a BoNT/A binder was experimentally observed to bind to the sameepitope as JDQ-B5 based on binding competition assays.

FIG. 5 shows a phylogenetic tree that compares the homology among BoNT/Abinding VHHs within the JDQ-B5 competition group to random alpaca VHHs.The homology comparison uses the unique amino acids that are presentbetween the forward PCR primer sequences and the hinge region (above).The distance of the lines is a measure of homology; the shorter thedistance separating two VHHs, the more homologous. Four VHHs that bindto the same epitope as JDQ-B5 cluster within a group that is distinctfrom the random VHHs as shown, strong evidence of relatatedness of theseclones. The results show that substantial variation in the VHH sequenceis tolerated without loss of the target binding capability.

The coding DNAs for two different VHH monomers were cloned in an E. coliexpression vector in several different ways to produce differentrecombinant proteins. To produce single VHH monomers, the VHH coding DNAwas inserted into the plasmid pET32b to fuse the VHH in frame with anamino terminal bacterial thioredoxin and a carboxyl terminal epitopictag (E-tag GAPVPYPDPLEPR; SEQ ID NO: 15). Additional coding DNA derivingfrom the pET32b expression vector DNA was also present between thethioredoxin and VHH coding sequences, the DNA encoding six histidines(to facilitate purification) and an enterokinase cleavage site, DDDDK topermit enzymatic separation of thioredoxin from the VHH. The resultingexpression vectors were used for expression of VHH monomers. VHHmonomers JDQ-H7 (SEQ ID NO: 32, referred to as “H7) and JDQ-B5 (SEQ IDNO: 24, referred to as “B5”) were expressed using this system (FIG. 6).A representation of the two monomer VHH proteins produced by theseexpression vectors, labeled H7/E and B5/E, is shown in FIG. 10 A.

Expression vectors were prepared in pET32b in which DNA encoding twoiterations of the VHH monomer (e.g., SEQ ID NOs: 46 and 48) was present,and the monomers joined in frame to yield heterodimers. For theseconstructions, the two nucleic acid sequences encoding the VHHs wereseparated by a nucleotide sequence encoding a 15 amino acid linker, SEQID NO: 55, that provides a flexible spacer (fs) between the expressedVHH proteins to separate the domains and facilitate independent folding.The E-tag coding DNA followed the second VHH coding DNA (SEQ ID NO: 49)in frame to obtain a single-tagged VHH heterodimer H7B5/E (SEQ ID NO:50), expression of which is shown in FIG. 10 B. A second copy of theE-tag coding DNA (e.g., SEQ ID NO: 51) was included upstream of thefirst VHH (at the amino coding end) for expression of a double-taggedVHH heterodimer E/H7/B5/E (SEQ ID NO: 52) shown in FIG. 10 B.

The thioredoxin fusion partner was included to improve expression andfolding of the VHHs, and was observed as not necessary for VHH function.The activity of the VHH agents to protect mice from BoNT/A intoxicationin mouse lethality assays was tested using VHH agents in whichthioredoxin was cleaved (by enterokinase) from the VHH. It was observedthat absence of thioredoxin caused no significant reduction in activity.

A single-tagged heterodimer VHH was predicted to lead to decoration ofthe BoNT toxin by the anti E-Tag mAb in a ratio of 1:1. Accordingly, asingle-tagged heterodimer was expected to bind at two sites on the toxinand lead to decoration of the toxin with two anti E-tag antibodies (seeFIG. 7). A double-tagged heterodimer provides for binding of the antiE-tag antibody in a ratio of 2:1 and thus should bind at two sites onthe toxin and lead to decoration of the toxin with four anti-tagantibodies (see FIG. 8). These agents were tested for their ability toprotect mice from BoNT/A toxin.

For these examples, the VHH agents and the toxin were pre-mixed and thenintravenously administered to groups of five subjects (mice) per group.The subjects were monitored and the time to death was noted for thosethat succumbed to the toxin. In the results shown in FIG. 9 A, a pool oftwo VHH monomers, H7/E and B5/E (1 μg of each monomer per subject), inthe presence of anti-E-tag mAb (Phadia, Sweden) (5 μg/mouse) delayeddeath only about one day in mice exposed to 1,000 LD₅₀ of BoNT/A. Thesingle-tagged VHH heterodimer, H7/B5/E (2 μg/mouse) in the presence ofanti-E-tag mAb (5 μg/mouse) delayed death by about a day and a half inmice exposed to 1,000 LD₅₀ of BoNT/A.

In contrast, it was observed that the double-tagged heterodimer,E/H7/B5/E (2 μg/mouse) administered with anti-E-tag mAb resulted in fullsurvival of mice exposed to 1,000 LD₅₀ and even 10,000 LD₅₀ of BoNT/A(FIG. 9B). Mice given the double-tagged VHH heterodimer, E/H7/B5/E, inthe absence of co-administered anti-E-tag mAb, did not survive a 1,000LD₅₀ amount of BoNT/E, showing that the anti-tag mAb was necessary forfull efficacy. The ability of the double-tagged VHH heterodimer,E/H7/B5/E, administered with anti-E-tag mAb to protect mice against10,000 LD₅₀ demonstrates that this treatment achieved a level ofefficacy similar to that obtained with a commercial polyclonal antitoxinsera.

In other examples, the BoNT/A-binding VHH heterodimer agents were testedfor their ability to prevent death in subjects previously exposed toBoNT/A. In these examples, groups of five subjects were first exposed to10 LD₅₀ BoNT/A. Then after 1.5 or three hours from exposure mice weretreated either with the E/H7/B5/E heterodimer agent (2 μg/subject)administered with anti-E-tag mAb (5 μg/subject), or with a dose ofpotent polyclonal anti-BoNT/A sera that had been prepared in sheep. Thissera had been previously shown to protect subjects against 10,000 LD₅₀of BoNT/A when it was co-administered with the toxin (assays performedas in previous paragraph). All subjects were monitored and the time todeath was determined for non-survivors. Control subjects (2 groups offive each) died within about a day. All subjects treated with polyclonalantisera 1.5 hour post-intoxication (five) survived, and four of fivesubjects treated three hours post-exposure both 1.5 hours and threehours post-intoxication survived. Five out of five subjects treated withthe VHH heterodimer and anti-E-tag mAb both 1.5 hours and three hourspost-exposure survived. Thus the VHH heterodimer and anti-E-tagtreatment was at least as effective as conventional polyclonalantitoxins at protecting subjects from BoNT exposure in the moreclinically relevant post-exposure challenge model.

Example. 12: Neutralization of Botulinum Neurotoxin Using VHH BindingProteins

Examples herein show that scFv antitoxin compositions preventdevelopment of disease symptoms in subjects exposed to a botulinumtoxin. These antitoxin agents were antibodies that bound the toxin andneutralized the activity of the toxin and/or promoted rapid clearancefrom the body. Data show that effective neutralization was achievedusing a mixture of two high-affinity toxin VHH binding agents, each ofwhich strongly neutralized toxin in cell-based assays. Administration ofa multimeric composition was much more effective at protecting subjectsfrom toxin than a pool of two neutralizing monomer binding agents only.

Camelid heavy chain only Vh domain (VHH) binding agents with highaffinity for Botulinum neurotoxin serotype A (BoNT/A) were producedincluding H7 (SEQ ID NO: 56), B5 (SEQ ID NO: 57). Methods of generatingVHH binding agents are shown in Shoemaker et al. U.S. application Ser.No. 12/032,744 which is application 2010/0278830 A1 published Nov. 4,2010, and Shoemaker et al. U.S. application Ser. No. 12/899,511 which isapplication 2011/0129474 A1 published Jun. 2, 2011, each of which isincorporated herein by reference in its entirety.

VHHs (H7, B5 and C2) displayed potent BoNT/A neutralization activity inassays of exposure or intoxication of primary neurons in culture. The H7VHH and B5 VHH monomers were expressed in E. coli and a singleheterodimeric polypeptide (H7/B5) was constructed and expressed with theH7 and B5 VHH domains/subunits separated by a fifteen amino acidflexible spacer having three repeats of amino acid sequence GGGGS (SEQID NO: 55). A combination of the H7 monomer binding agent and B5 monomerbinding, and a H7/B5 single chain heterodimer binding agent were testedto determine ability to protect mouse subjects from death caused byBoNT/A. The subjects received ten-fold the lethal dose of BoNT/A thatcauses death in 50% of mice (10 LD₅₀), and either 1.5 hours or threehours later were administered either: 1 micrograms (μg) of H7 bindingagent; a sheep antitoxin serum produced against BoNT/A; 1 of B5 monomerbinding agent; or 2 μg of H7/B5 single chain heterodimer binding agent(FIG. 11 A-B). The amount of sheep antitoxin serum administered wasequivalent to the amount of commercial antitoxin serum generallyadministered.

Data show that subjects administered a combination of monomeric H7 andB5 binding agents died within three days. Control subjects administeredno therapeutic agent died within one day (FIG. 11 A-B). Subjectsadministered the sheep antitoxin serum survived at 80%. Most important,subjects administered H7/B5 single chain heterodimer binding agentsurvived additional days compared to the control subjects, with 80% ofsubjects administered H7/B5 heterodimer binding agent surviving forseven days.

Example. 13: Neutralization of C. Difficile Toxins Using HeteromultimerBinding Agents

A set of VHH binding agents that bind Clostridium difficile toxin B(TcdB) were obtained and shown in Examples herein to inhibit the abilityof the toxin to intoxicate or infect cells (FIG. 12 A). Potent anti-TcdBneutralizing VHHs were selected, identified by codes names 5D and E3,and were expressed as separate monomers or as a heterodimer. Apool/mixture of VHH monomers, 5D and E3, was compared in for ability toprevent TcdB lethality to cells to the 5D/E3 heterodimer.

CT26 cells were exposed to TcdB (100 picograms/ml) in the presence ofdifferent concentrations (0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, or30 nM) of: a mixture of 5D VHH monomer (SEQ ID NO: 67) and E3 VHHmonomer (SEQ ID NO: 68), or a 5D/E3 heterodimer (SEQ ID NO: 87). Controlcells were not administered neutralizing agents. Cell rounding caused byTcdB was monitored using a phase-contrast microscope.

Culture media from expressing cells were administered with either themixture of 5D and E3 VHH monomers, or the 5D/E3 VHH heterodimer werefound to be effective in protecting the cells from TcdB associated cellrounding. Control cells (100%) showed cell rounding and negative indiciaof TcdB following toxin exposure.

It was observed that administering 0.1 nM 5D/E3 heterodimer to subjectsprior to TcdB exposure resulted in 50% cell rounding (i.e., 50% TcdBinfection; FIG. 12 B). The same level cell rounding protection (50%),was achieved with 1 nM of the mixture of 5D and E3 monomers. Thus, the5D/E3 VHH heterodimer was observed to be about ten-fold more potent as atoxin neutralizing agent than a pool containing the same two VHHs asmonomers (FIG. 12 B).

The improved antitoxin and protective potency 5D/E3 heterodimer wasfurther analyzed using an in vivo toxin challenge mouse model. Subjectswere co-administered a lethal dose of TcdB (1 ng/mL) with either amixture of 500 nanograms (ng) of 5D monomer and 500 ng E3 VHH monomer;or with 250 ng of 5D/E3 VHH heterodimer; or with phosphate bufferedsaline, PBS. See FIG. 12 C. See Data show that each of the VHH bindingagents was a more effective TcdB neutralizing agent for subjects thanthe PBS control. Survival was observed at 100% for subjects administered5D/E3 VHH heterodimer (250 ng) and at about 40% for subjectsadministered a mixture of 5D and E3 VHH monomers. Control subjectsreceiving PBS survived at a rate of 20%.

Data show that subjects administered a mixture of 5D and E3 monomerssurvived for fewer days and were less protected from a lethal TcdBchallenge than subjects administered the 5D/E3 heterodimer (FIG. 12 C).Most important the improved protection and neutralizing ability of the5D/E3 heterodimers was observed even if the amount of heterodimeradministered was 75% less than the amount of the mixture of 5D and E3monomers. Further analysis was performed in Examples below to determinethe relative factors for VHH monomers and heterodimers to effectivelyneutralize and clear disease agent targets from the body (FIG. 12 A-C).

Example 14: Identification and Characterization of Anti-BoNT VHHs

Serum clearance of Botulinum neurotoxin serotype A (BoNT/A) wasdramatically accelerated by administering a pool of differentepitopically-tagged single-chain Ig variable fragment (scFv) domainbinding agents with an anti-tag monoclonal antibody (Shoemaker et al.U.S. Ser. No. 12/032,744 application 2010/0278830 A1 published Nov. 4,2010; Shoemaker et al. U.S. Ser. No. 12/899,511 application 2011/0129474A1 published Jun. 2, 2011; Sepulveda et al. 2009 Infect Immun 78:756-763, and Tremblay et al. 2010 Toxicon. 56(6): 990-998, each of whichis incorporated herein in its entirety).

To determine whether a more commercially and clinically acceptablebinding agent than scFvs could be identified, a panel of camelidheavy-chain-only Vh (VHH) binding agents having high affinity forepitopes of BoNT/A holotoxin was produced. VHHs were obtained that boundto an epitope of a distinct BoNT serotype, BoNT/B holotoxin, and theseVHHs were tested for antitoxin efficacy. Competition ELISAs wereperformed to identify the VHHs with the highest affinity for uniqueepitopes on BoNT/A and BoNT/B. VHHs specific for each of BoNT/A (FIG. 13A) and for BoNT/B (FIG. 13 B) were identified.

The VHHs in FIG. 13 A-B include amino acid sequence QLQLVE (SEQ ID NO:88) and QVQLVE (SEQ ID NO: 89) at the amino terminus region. Thesequence was encoded by the PCR primer used to generate the VHH-displaylibrary (Maass et al. 2007 Int J Parasitol 37: 953-962). The eight aminoacids shown at the carboxy-terminus end were encoded by the short hingeor long hinge PCR primers that were used to generate the VHH library.

The amino acid sequences for double-tagged VHH heterodimer antitoxinsthat specifically bind BoNT/A: ciA-H7/ciA-B5(2E) and ciA-F12/ciA-D12(2E)are shown in FIG. 13 C. Each heterodimer included two VHH monomers andtwo epitopic tags. The amino acid sequences of the tags within the aminoacid sequences of the heterodimers are underlined (FIG. 13 C). The aminoacid sequence preceding the first E-tag in each VHH protein containedthe thioredoxin fusion partner and hexahistidine encoded by the pET32bexpression vector. The VHH sequences were flanked by the two E-tagpeptides and were separated by the unstructured spacer having amino acidsequence (GGGGS)₃, SEQ ID NO: 55.

Each VHH was purified from E. coli as a thioredoxin fusion proteincontaining a single carboxyl-terminal epitopic tag (E-tag). Sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysesof VHH monomers and VHH heterodimers was performed (FIG. 14 A-B). Thechannels were loaded with one microgram (μg) of each VHH monomer orheterodimer. Molecular weight markers (12, 31, 45, 66, 97, 116 and 200kilodaltons) are shown on the border of each gel. FIG. 14 A showsSDS-PAGE analysis of the tagged (E) VHH monomers: ciA-D1, ciA-H4,ciA-H11, ciA-A5, ciA-C2, ciA-D12, ciA-F12, ciA-G5, and ciA-H7. Darkbands were observed at approximately 35-38 kilodalton molecular weightfor all single tagged VHH monomers. Channels loaded with ciA-D1, ciA-H4,ciA-H11, and ciA-B5 showed light bands at about 45-46 kilodaltons (kDa),and at about 59 kDa to about 62 kDa molecular weight. SDS-PAGE analysiswas performed also on single- or double-tagged VHH heterodimers:ciA-H7/ciA-B5 singly tagged on ciA-B5; double tagged ciA-H7/ciA-B5having a tag on both ciA/H7 and ciA-B5, ciA-F12/ciA-D12 singly tagged onciA-B5; double tagged ciA-F12/ciA-D12 having a tag on both ciA/F12 andciA-D12, double tagged ciA-A1 1/ciA-B5 having a tag on both ciA/A11 andciA-B5 (FIG. 14 B). Strong dark bands at about 48 kDa to about 58 kDamolecular weight were observed for each heterodimer (FIG. 14 B).

The unique BoNT/A binding VHHs were further characterized and analyzedfor ability to affinity target BoNT/A using surface plasmon resonance(SPR) in which a lower Kd indicates stronger binding/affinity betweenthe VHH and the toxin target. Analysis was performed also to determinethe ability of the BoNT/A binding VHHs to prevent intoxication ofprimary neurons in culture (FIG. 15 and Table 5).

Neuronal granule cells from pooled cerebella of seven day old to eightday old Sprague-Dawley rats or five day old to seven day old CD-1 micewere harvested as described by Skaper et al 1979 Dev Neurosci 2:233-237.The cells were then cultured in 24-well plates as described by Eubankset al 2010 ACS Med Chem Lett 1: 268-272. After a week or more ofculture, each culture well was adjusted to a volume of 0.5 ml withdilutions of VHHs (ciA-H7, ciA-B5, ciA-C2, ciA-D12, ciA-F12, ciA-A5 orciA-G5) or a buffer control, and BoNT/A (ten picomoles) was added. Afterovernight incubation at 37° C., cells were harvested and the extent ofsynaptosomal-associated protein 25 (SNAP25) cleavage was determined byWestern blot using commercially available rabbit anti-SNAP25(Sigma-Aldrich Inc.). See FIG. 15. SNAP-25 is a membrane bound proteinanchored to the cytosolic face of membranes by palmitoyl side chainswithin the molecule that is involved in the regulation ofneurotransmitter release. Botulinum toxin serotypes including serotypesA, C and E function to cleave SNAP-25, resulting in paralysis andclinically developed botulism.

The upper band shown in the Western blot photographs is uncleavedSNAP25, and the lower band indicates cleaved SNAP25 (FIG. 15). SNAP25cleavage (i.e., presence of a lower band) resulting from exposure tobotulinum toxin was observed. VHHs were identified by the criterion thatat concentrations of less than 0.1 nanomoles (nM) were observed toinhibit BoNT/A cleavage of SNAP25 (i.e., no lower band), are strongneutralizing agents. Weak neutralizing VHHs were identified as VHHs thatrequired greater than 1 nM to inhibit BoNT/A cleavage of SNAP25. VHHsthat required greater than 10 nM to prevent SNAP25 cleavage wereidentified as having no toxin neutralizing ability (FIG. 15).

It was observed that about equimolar amounts of ciA-B5, ciA-C2 andciA-H7 VHHs prevented intoxication of neurons with 10 picomoles ofBoNT/A. Two VHHs (ciA-D12 and ciA-F12) were observed to have no ornegligible toxin neutralizing activity even at 1,000-fold excess VHH totoxin. Two VHHs (ciA-A5 and G5) displayed intermediate neutralizingactivity compared to ciA-B5, ciA-C2 and ciA-H7, the stronglyneutralizing VHHs, and ciA-D12 and ciA-F12, the non-neutralizing VHHs(FIG. 15 and Table 5).

Thus, ciA-B5, ciA-C2 and ciA-H7 were determined to be strongneutralizing VHHs. Other isolates including ciA-D12 and ciA-F12 wereobserved to be non-neutralizing VHHs that produced no detectable toxinneutralization.

Example 15: Protection from BoNT/a Lethality Using Monomeric Anti-BoNT/aVHHs

Epitopically tagged anti-BoNT/A VHH compositions were shown in theExample herein to prevent toxin induced lethality in the presence orabsence of the clearing anti-tag mAb. Methods of testing VHHs are shownin Sepulveda et al. 2009 Infect Immun 78:756-763, and Tremblay et al.2010 Toxicon. 56(6): 990-998. Pools/mixtures of two, three, four or sixdifferent anti-BoNT/A VHH monomers with or without anti-E-tag clearingantibody were co-administered to subjects with an amount (1000 LD₅₀ or10,000 LD₅₀) of BoNT/A holotoxin. Subjects were then monitored forsymptoms of toxin lethality and were observed for time to death.

The subjects were co-administered BoNT/A with either a mixture of ciA-H7and ciA-B5 monomers, or a mixture of ciA-D12 and ciA-F12 monomers (FIG.16 A bottom graphs). Each mixture was administered with (+αE) or without(−αE) anti-E-tag clearing antibody that specifically bound the epitopictags located on the VHHs. Control subjects were administered toxin only.Unless indicated otherwise, a dashed line in FIGS. 16-24 indicates thatno anti-E-tag antibody was administered to the subjects. Each monomericVHH was used at a total dose of two micrograms (μg) per mouse to ensurethat the only the complexity and/or identity of the VHH mixtures wasvaried among groups and was the cause of observed antitoxin efficacy.

Results obtained show that subjects administered ciA-D12 and ciA-F12,two anti-BoNT/A VHH monomers previously determined not to neutralizeBoNT/A in cell assays, did not survive toxin challenge for any greatertime than did control subjects administered toxin only (FIG. 16 A bottomgraphs). Administration of 5 μg amounts of anti-E-tag clearing antibody(αE) to subjects only slightly prolonged time before death. Data showthat subjects administered neutralizing VHH monomers ciA-H7 and ciA-B5with anti-E-tag clearing antibody were slightly protected against BoNT/Acompared to subjects administered ciA-D12 and ciA-F12, and anti-E-tagclearing antibodies. Thus, the decoration of BoNT/A with two clearingantibodies provided little or no therapeutic benefit to the subjects.

Administration to subjects of a mixture of ciA-B5, and ciA-H7 monomersabsent clearing antibody only delayed time to death. Data show thatsubjects challenged with 100-fold the LD₅₀ of BoNT/A (approximately 5nanograms total) survived longer following administration of a mixtureof neutralizing ciA-B5 and ciA-H7 compared to control subjectsadministered no VHHs. Most important, it was observed thatco-administration of clearing antibody and the neutralizing VHHsresulted in 100% survival of subjects challenged with 100-fold the LD₅₀of BoNT/A (FIG. 16 A bottom left graph). At a challenge of 1,000-foldthe LD₅₀ of BoNT/A, death was delayed about one additional day forsubjects co-administered a mixture of ciA-B5 and ciA-H7 and anti-E-tagclearing antibody compared to subjects administered VHHs alone orcontrol subjects (FIG. 16 A bottom right graph). Thus, it was observedthat administering a mixture of toxin neutralization VHH monomers withclearing antibody provided greater therapeutic benefit and protectionagainst BoNT/A than administering VHHs absent the clearing antibody.Relative affinity of each VHH influences the therapeutic effect of theVHH, likewise for VHHs having similar sub-nanomolar affinities (SeeTable 5).

Whether mixtures of VHH monomers containing both neutralizing VHHs andnon-neutralizing VHHs were effective antitoxin agents was furthertested. Subjects were co-administered 1,000-fold or 10,000-fold BoNT/ALD₅₀ and one VHH monomer mixture of either a mixture of ciA-B5, ciA-H7,and ciA-C2; or a mixture of ciA-H7, ciA-A5 and ciA-D12 with (+αE) orwithout (−αE) an anti-E-tag clearing antibody preparation thatspecifically binds the epitopic tags located on the VHHs (FIG. 16 Bbottom graphs). Control subjects were administered toxin only.

Administration of a mixture of ciA-B5, ciA-H7, ciA-C2 monomers, eachcapable of potent toxin neutralization, delayed death less than a day inmice exposed to 1000-fold the LD₅₀ of BoNT/A (FIG. 16 B bottom leftgraph). Subjects were completely protected (100% survival) at 1000-foldthe LD₅₀ of BoNT/A following administration mixture of ciA-B5, ciA-H7,and ciA-C2 monomers and clearing antibody. Co-administration of10,000-fold the LD₅₀ of BoNT/A (a total amount of 0.5 μg), a mixture ofciA-B5, ciA-H7, ciA-C2 monomers and clearing antibody delayed death morethan two days in subjects (See FIG. 16 B bottom right graph) compared tocontrol subjects.

It was observed that administration of a mixture of ciA-H7, ciA-A5, andciA-D12 in which two VHH monomers (ciA-A5 and ciA-D12) in the mixture ofmonomers were weak toxin neutralizers, resulted in subjects survivingmuch less after exposure to an amount of BoNT/A 1,000-fold BoNT/A LD₅₀(FIG. 16 B bottom left graph).

Thus, administration of the mixture of ciA-B5, ciA-H7, and ciA-C2 taggedmonomers, each of which are strong neutralizing VHHs, to subjectsprovided greater protection against BoNT/A than the mixture of ciA-H7,ciA-A5 and ciA-D12, in which two of the three VHH monomers do notneutralize BoNT/A. Data show that 100% of subjects administered themixture of ciA-B5, ciA-H7, and ciA-C2 with the anti-tag clearingantibody survived a dose of BoNT/A that was 1,000-fold the LD₅₀ of aBoNT/A (FIG. 16 B bottom left graph), and survived additional daysfollowing administration of 10,000-fold the LD₅₀ of a BoNT/A (FIG. 16 Bbottom left graph).

Table 5: SPR binding data for VHH monomers and heterodimers

TABLE 5 SPR binding data for VHH monomers and heterodimers SPR Kd Cloneprotein epitope^(#) neutralization* (nM) subunit{circumflex over ( )}Genbank JDY-33 ciA-H7 A1 strong 0.06 +/− 0.07 Lc HQ700708 JDT-2 ciA-D1A1 strong  0.71 +/− 0.004 Lc JEC-3 ciA-H4 A1 not done 1.54 +/− 0.06 LcJEC-11 ciA-H1 A1 not done  4.3 +/− 0.09 Lc JDY-46 ciA-C2 A2 strong 2.7+/− 3.1 Lc HQ700705 JDY-9 ciA-B5 A3 strong 0.17 +/− 0.06 Hc HQ700704JED-27 ciA-F12 A4 none 0.24 +/− 0.03 Lc HQ700706 JDU-26 ciA-D12 A5 none0.21 +/− 0.1  Lc HQ700702 JDY-2 ciA-A5 A6 weak 1.05 +/− 0.05 noneHQ700703 JDY-59 ciA-G5 A7 weak 0.32 +/− 0.03 none HQ700707 JFA-10ciB-H11 B1 not done 0.26 +/− 0.01 none JFX-30 ciB-A11 B2 not done 0.84+/− 0.68 none JFV-48 ciB-B5 B3 not done 0.97 +/− 0.04 none JFV-40 ciA-B9B4 not done 23 +/− 5.6 none JEZ-2 ciA-H7/B5 A1/A3 strong 0.014 +/− 0.007not done JFK-21 ciA-F12/D12 A4/A5 not done 0.097 +/− 0.038 not doneJGA-3 ciB-A11/B5 B2/B3 not done 5.3 +/− 1.5 not done

Complete survival (100%) was observed for subjects administered amixture of ciA-B5, ciA-H7, ciA-D12 and ciA-F12 tagged monomers andanti-tag clearing antibodies of the challenge with an amount of BoNT/Athat was 1,000-fold the LD₅₀ (FIG. 16 C bottom left graph).Administering a pool of anti-BoNT/A VHHs (ciA-B5, ciA-H7, ciA-D12 andciA-F12) in which only two VHHs (ciA-B5, ciA-H7) were strong toxinneutralizers only slightly delayed death in subjects exposed to1000-fold the LD₅₀ of BoNT/A (FIG. 16 C bottom left graph). At10,000-fold the LD₅₀ of a BoNT/A, subjects co-administered the mixtureof four VHH tagged monomers and anti-tag clearing antibody survivedadditional days compared to control subjects (FIG. 16 C bottom leftgraph).

The antitoxin efficacy of a pool of four anti-BoNT/A VHHs taggedmonomers (ciA-A5, ciA-B5, ciA-C2 and ciA-H7) was compared to a pool ofsix different VHH tagged monomers (ciA-A5, ciA-B5, ciA-C2, ciA-H7,ciA-D12, and ciA-G5). The pool of six VHH monomers contained the sameVHHs as the pool of four VHHs and further included two VHHs (ciA-D12,and ciA-G5) that were weak neutralizers of BoNT/A (FIG. 17 and Table 5).The different pools of VHH monomers were each administered in thepresence of clearing anti-tag antibody. It was observed that 100% ofsubjects administered either the pool of four VHH tagged monomers or thepool of six VHHs tagged monomers with anti-tag clearing antibodysurvived challenge with 1000-fold the LD₅₀ of BoNT/A (FIG. 17 leftgraph). Subjects challenged with 10,000-fold the LD₅₀ of BoNT/A survivedlonger following co-administration of either the pool of four VHHmonomers or the pool of six VHH monomers with clearing anti-tagantibody, than control subjects administered only toxin (FIG. 17 rightgraph). These results show that decoration of BoNT/A with a greaternumber of VHH antibodies, four or more VHHs, greatly improved antitoxinefficacy. Administering a pool of four VHH monomers or a pool of six VHHmonomers to the subjects provided additional antitoxin efficacy comparedto administering three or fewer VHH monomers.

These data clearly show that toxin clearance was rendered much moreeffective under conditions in which BoNT is decorated by at least threeVHH antibodies and at least about three clearing antibodies. It wasobserved also that mixtures of monomers having greater number orpercentage of toxin neutralization VHHs greatly contributed to percentsurvival of subjects co-administered a vast excess of the lethal dose ofBoNT/A.

Example 16: VHH Affinity and Antitoxin Efficacy

Toxin neutralization and clearance mechanisms were observed herein todepend on affinity of antitoxin binding to the toxin. Without beinglimited by a particular theory or mechanism of action, the kinetics oftoxin binding (K_(on)) and release (K_(off)) by the antitoxin bindingagents contribute to the antitoxin efficacy.

To determine the relationship of toxin affinity to antitoxin efficacyand the role of each, assays were performed for identification ofmultiple VHHs recognizing the same epitope. In the course of anti-BoNT/AVHH screening and based on competition ELISA analysis, several VHHs(ciA-D1, ciA-H4 and ciA-H11) were identified that recognized the sameepitope as ciA-H7. SPR analysis showed that each VHH monomer recognizedand bound the ciA-H7 epitope with a different affinity. The dissociationconstant (Kd) identifies the strength of binding or affinity between aligand and a receptor, between the VHH antibody and the toxin.

The VHH Kd values for the VHHs having the stronger binding to BoNT/Awere determined to be 0.06±0.07 nM for ciA-H7, 0.71±0.004 for ciA-D1,and the VHH Kd values for the VHHs having the weakest binding to BoNT/Awere determined to be the 1.54±0.06 for ciA-H4, and 4.3±0.09 for ciA-H11respectively (FIG. 18 A). These four VHHs were tested with anti-tagclearing antibody for their efficacy as antitoxin VHHs in combinationwith the two VHHs (ciA-B5, ciA-C2) that recognize distinct,non-overlapping epitopes of BoNT/A (FIG. 18 B left and right graphs).

Subjects (five mice per group) were co-administered BoNT/A and one offour mixtures containing three VHH monomers: ciA-H7, ciA-B5 and ciA-C2;ciA-D1, ciA-B5 and ciA-C2; ciA-H4, ciA-B5 and ciA-C2; or ciA-H11, ciA-B5and ciA-C2. Each mixture included two strong neutralizing VHH monomers(ciA-B5 and ciA-C2), and one VHH of ciA-H7, ciA-D1, ciA-H4, or ciA-H11.Control subjects received toxin only.

Data show that 100% of subjects survived following co-administration of100 BoNT/A LD₅₀ and VHH mixtures containing ciA-B5 and ciA-C2 and eitherciA-H7, ciA-D1 or ciA-H4. Subjects administered the VHH mixture ofciA-B5, ciA-C2 and ciA-H11 survived the 100 LD₅₀ of BoNT/A at 80% (FIG.18 B left graph). Among subjects challenged with 1,000-fold the LD₅₀ ofa BoNT/A (FIG. 18 B right graph), the level of protection was a functionof the relative binding affinity or Kd of the VHH to BoNT/A shown inFIG. 18 A. Specifically the greatest protection at 1,000-fold BoNT/ALD₅₀ was observed in subjects administered the VHH mixture containingciA-B5, ciA-C2, and ciA-H7, which had the strongest BoNT/A affinity(i.e., lowest Kd value of 0.06±0.07; FIG. 18 A and FIG. 18 B rightgraph). The least extent of protection was observed in subjectsadministered the VHH mixture containing ciA-B5, ciA-C2, and ciA-H11(weakest BoNT/A affinity and highest Kd value of 4.3±0.09; FIG. 18 A andFIG. 18 B right graph), the survival of which was comparable to controlsubjects not administered VHH monomers.

Correlating the Kd values with antitoxin-toxin binding and affinities,it was observed that the lower the Kd value the greater the respectivetoxin affinity and the greater the antitoxin efficacy of the VHH. VHHciA-H7 was observed to have the lowest Kd and the strongest bindingaffinity to BoNT/A, and was determined to have greater antitoxinefficacy than other VHH compositions identified in FIG. 18 A. Thus,sub-nanomolar affinities or Kd values for the tagged toxin bindingagents is an important factor in identifying the VHH with greatestantitoxin efficacy and most effective ability to protect subjects fromtoxin-associated infection and death.

Example 17: Antitoxin VHHs Heterodimers

By engineering and expressing two anti-BoNT/A VHHs as a heterodimer, aresulting multimeric binding protein molecule was obtained, and thiscomposition was found to bind to two different sites on the toxin andyield an improved toxin affinity. Examples herein analyzed the role ofepitopic tags on the heterodimer and the role of the amount of thetagging of the heterodimer compared to the clearing antibody withrespect to increasing antitoxin efficacy of the heterodimer.

VHH heterodimers were engineered to contain an epitopic tag fordecoration of BoNT/A with two anti-tag clearing antibodies (FIG. 19 Atop drawing). Survival and protection of subjects was analyzed followingchallenge with each of 100-fold and 1000-fold the LD₅₀ of BoNT/A (FIG.19 A bottom left and right graphs). Data show that administeringheterodimer containing two strongly neutralizing VHHs, ciA-B5 andciA-H7, resulted in greater antitoxin efficacy and longer survival ofsubjects than administering heterodimers containing two weak ornon-neutralizing VHHs, ciA-D12 and ciA-F12 (FIG. 19 A bottom left andright graphs).

A second copy of the epitopic tag to the heterodimers compared to onlyone epitopic tag was observed to promote toxin decoration with fourclearing antibodies and to yield greater clearing efficacy (FIG. 19 Btop drawing). All (100%) of subjects survived a challenge with either1000-fold or 10,000-fold the LD₅₀ of BoNT/A and co-administration ofciA-B5/ciA-H7 heterodimer having two epitopic tags and anti-tag clearingantibody (FIG. 19 B bottom graphs).

To further analyze whether two or more epitopic tags improvedheterodimer antitoxin efficacy, two sets of anti-BoNT/A VHH heterodimerswere constructed in which the two VHHs in the heterodimers were eithernon-neutralizing (ciA-D12/F12) or potent toxin neutralizing agents(ciA-B5/H7). The two different VHH heterodimers were engineeredcontaining either one or two copies of the epitopic tag (E-tag) and wereexpressed. SPR analysis confirmed that the heterodimer affinities werein the range of 10 picomolar to 100 picomolar which was significantlygreater than the affinities of the component monomers (FIG. 15 and Table5).

The antitoxin efficacies of the single tagged heterodimers administeredto mouse subjects after challenge with 1000-fold LD₅₀ of BoNT/A (FIG. 19A bottom left graph) were observed to be similar to results obtainedfrom administering a mixture of the two corresponding monomers only(FIG. 16 A bottom right graph). Administering the non-neutralizingsingle-tagged heterodimer, ciA-D12/F12(1E), resulted in no protectionfrom challenge with 1000-fold LD₅₀ of BoNT/A in the absence of clearingantibody, and only slightly delayed death in the presence of clearingantibody 9 FIG. 19 A bottom left graph). The toxin neutralizingsingle-tagged heterodimer, ciA-B5/H7(1E), delayed death in mice exposedto 1000 LD₅₀ BoNT/A for one to two days in the absence of clearingantibody and efficacy was only slightly improved by the addition ofclearing antibody (FIG. 19 A bottom left graph).

Improved antitoxin efficacy was observed in subjects administered aheteromultimeric agent having a second copy of the epitopic tag, withboth non-neutralizing and neutralizing anti-BoNT/A VHH heterodimers inwhich the heterodimer agent was co-administered with clearing antibody.Without being limited by any particular theory or mechanism of action,it is here envisioned that component binding regions in a ‘double-taggedheterodimer’ bind at two sites on the toxin and each bound heterodimerdecorates toxin with two clearing antibodies, resulting in decoration ofthe toxin with at least four clearing antibodies (FIG. 19 B top drawing)which Examples herein show had increased clearance. Administeringnon-neutralizing double-tagged heterodimer containing ciA-D12/F12(2E)resulted in virtually no antitoxin efficacy in subjects in the absenceof clearing antibody at both 1000-fold and 10,000-fold the LD₅₀ ofBoNT/A (FIG. 19 B bottom left and right graphs). In the presence ofclearing antibody, ciA-D12/F12(2E) heterodimer fully protected subjects(100% survival) from 100-fold BoNT/A LD₅₀ and delayed death about a dayin subjects receiving 1000-fold BoNT/A LD₅₀ compared to control subjects(FIG. 19 B bottom right graph and FIG. 20 left graph). Thus the presenceof a second epitopic tag attached to the heterodimer dramaticallyimproved the antitoxin efficacy.

Non-neutralizing heterodimer, ciA-D12/F12, with either one, two or threeepitopic tags was analyzed for antitoxin efficacy in the presence ofclearing antibody (FIG. 20). The single-tagged heterodimer only slightlyprotected subjects from toxin challenge of 100-fold the LD₅₀ of BoNT/A.Subjects challenged with double-tagged heterodimers and triple-taggedheterodimers were fully protected from a challenge of 100-fold the LD₅₀of BoNT/A (FIG. 20 left graph). Only little improvement in antitoxinefficacy was observed with the triple-tagged heterodimers compared tothe double-tagged heterodimers, consistent with the observation thatnear maximal clearance was achieved by decorating the target with fourclearing antibodies. A titration of the clearing antibody administeredwith the double-tagged ciA-D12/F12 heterodimer demonstrated that maximalantitoxin efficacy against both 100-fold and 1,000-fold the LD₅₀ ofBoNT/A was achieved with the number of clearing antibody molecules(measured in picomoles) administered in an amount approximatelyequivalent to the number of epitopic tags (FIG. 21 left and rightgraphs).

An even more dramatic antitoxin effect was observed in cell cultureintoxication assays using the double-tagged heterodimer, ciA-B5/H7(2E),in which both of the component anti-BoNT/A VHHs individually possesspotent neutralizing activity (FIG. 15). In the absence of clearingantibody, the double-tagged ciA-B5/H7(2E) heterodimer produced the sameantitoxin efficacy as the equivalent single-tagged heterodimer (compareFIG. 19 A bottom left and right graphs to FIG. 19 B bottom left andright graphs). In the presence of clearing antibody, the neutralizingdouble-tagged heterodimer at 40 picomoles (pmoles) was observed to be ahighly potent antitoxin that fully protected cells from lethality whenco-administered with 10,000-fold the LD₅₀ of BoNT/A, i.e., the totalamount was about 3 pmoles.

A dose-response assay was performed in mouse subjects with double-taggedciA-B5/H7(2E) heterodimer co-administered with 1000-fold the LD₅₀ ofBoNT/A (FIG. 22). It was observed that both 40 pmoles and 13 pmoles ofdouble-tagged ciA-B5/H7(2E) heterodimer completely protected thesubjects against an exposure of 1000-fold the LD₅₀ of BoNT/A. A dose of4 pmoles ciA-B5/H7(2E) heterodimer had the same protective efficacy for1,000-fold the LD₅₀ of BoNT/A as a dose of 40 pmoles did with10,000-fold the LD₅₀ of BoNT/A (FIG. 15 B and FIG. 22). These data showthat co-administering about a fifteen-fold molar excess of thedouble-tagged heterodimer binding agent with the clearing antibody wassufficient to effectively neutralize and/or clear substantially all(greater than 99.99%) of the BoNT/A.

Example 18: Recombinant Antitoxin Efficacy in a Clinically RelevantPost-Intoxication Assay

Assays in which varying doses of toxins are co-administered withantitoxin agents were observed to permit sensitive quantification ofantitoxin efficacy. To more accurately reflect the typical clinicalsituation, antitoxin agents were tested in an assay of greater clinicalrelevance by intraperitoneally administering to mouse subjects ten-foldthe LD₅₀ of BoNT/A, and at 1.5 hours and three hours afterwards,administering intravenously neutralizing heterodimer antitoxin agentswith and without the anti-tag clearing antibody. Different sets ofanti-BoNT/A VHH heterodimers were tested: a heterodimer containingnon-neutralizing double-tagged ciA-D12/F12(2E), and a heterodimercontaining neutralizing double-tagged ciA-H7/B5(2E) heterodimer (FIG. 23A-B). A potent sheep anti-BoNT/A serum was used as a control in theassay at a dose demonstrated to protect 100% of mice from lethalitygiven 10,000-fold the LD₅₀ of BoNT/A.

The non-neutralizing ciA-D12/F12(2E) heterodimer was observed to havelittle or no antitoxin efficacy in absence of clearing antibodyfollowing administration either 1.5 hours or three hours after BoNT/Achallenge (FIG. 23 A left and right graphs). However, ciA-D12/F12(2E)heterodimer administered with clearing antibody displayed an efficacynearly equivalent to the positive control sheep antiserum (FIG. 23 Bleft and right graphs). These results show that toxin clearance alone issufficient to protect mice from a low dose BoNT challenge, even whenadministered several hours post-exposure to toxin.

Surprisingly the neutralizing ciA-H7/B5(2E) heterodimer was observed tobe as highly effective as an antitoxin in this assay, in the presence oreven absence of clearing antibody (FIG. 23 B). The double-tagged toxinneutralizing heterodimer administered 1.5 hours after toxin challengewith ten-fold the LD₅₀ of BoNT/A resulted in an antitoxin efficacyequivalent to the sheep serum polyclonal antitoxin. It was observed thatfollowing challenge at 10 BoNT/A LD₅₀ for 1.5 hours, subjectsadministered ciA-H7/B5(2E) heterodimer absent anti-tag clearing fullysurvived (100% survival; FIG. 23 B left graph). The survival forsubjects administered ciA-H7/B5(2E) heterodimer was comparable tosubjects administered sheep antitoxin (FIG. 23 B left graph).

Data show that three hours after toxin challenge at ten-fold the LD₅₀ ofBoNT/A, the neutralizing ciA-H7/B5(2E) heterodimer resulted in greatersubject survival (80%) than the sheep serum polyclonal antitoxin (60%survival; FIG. 23 B right graph). Most important, the survival ofsubjects using neutralizing ciA-H7/B5(2E) heterodimer was the same withor without clearing antibody (FIG. 23 B right graph).

These data clearly show that BoNT neutralization was sufficient for fullantitoxin efficacy in a clinically relevant post-intoxication(post-exposure to toxin) assay with low dose toxin challenge. A singlerecombinant multimeric binding protein with potent toxin neutralizationproperties was as effective as antitoxin sera in a model of a typicalclinical situation involving toxin exposure and subsequent treatment.

Example 19: Antitoxin Efficacy of a Double-Tagged Heterodimer TargetingBotulinum Toxin, BoNT/B

Double-tagged VHH heterodimer antitoxins that specifically recognizedand bound unique epitopes on BoNT/B holotoxin (FIG. 13 B) wereidentified and expressed. Two of the VHHs, ciB-A11 and ciA-B5, wereobserved to be the most effective antitoxins of those obtained frommonomer pool assays, and were engineered and expressed as double-taggedheterodimer, ciB-A11/B5(2E).

Subjects were exposed to either 1,000-fold (FIG. 24 A left graph) or10,000-fold (FIG. 24 A right graph) BoNT/B LD₅₀, and were administered aciB-A11 and ciB-B5 heterodimer with (+αE) or without (−αE) anti-tagclearing antibody. Control subject were exposed only to toxin (noheterodimer binding proteins). Data show that in the presence ofclearing antibody the ciB-A11/B5(2E) heterodimer fully protectedsubjects challenged with 1000-fold the LD₅₀ of BoNT/B (FIG. 24 A leftgraph) and extended the life of subjects challenged with 10,000-fold theLD₅₀ of BoNT/B (FIG. 24 A right graph).

Analysis was performed to determine whether the ciB-A11 and ciA-B5double tagged heterodimer was effective to treat subjects in a BoNT/Bpost-exposure in vivo model.

Subjects were intravenously exposed to 10 LD₅₀ of BoNT/A, and thenadministered 1.5 hours or three hours afterward either: ciB-A11 andciA-B5 double tagged heterodimeric protein with or without clearingantibody, or a sheep antitoxin serum. Control subjects were only exposedto 10 LD₅₀ of BoNT/B (no heterodimeric binding protein wasadministered). See FIG. 24 B left and right graphs. Data show 60% ofsubjects administered ciB-A11/B5 double tagged heterodimer with anti-tagantibody survived 1.5 hours and three hours after BoNT/B exposure, andfurther that 20% more subjects survived with ciB-A11/B5 double taggedheterodimer with clearing antibody treatment than with sheep antitoxinat both time points (FIG. 24 B left and right graphs). It was observedthat three hours after BoNT/B exposure subjects administered A11/B5double tagged heterodimer binding protein only (without anti-tagantibody) survived as long as subjects administered sheep antitoxin(FIG. 24 B right graph).

Results from these clinically relevant post-intoxication assays hereinshowed that ciB-A11/B5 heterodimer with or without clearing antibody wasas effective as sheep anti-BoNT/B serum in protecting subjects fromdeath caused by BoNT/B holotoxin exposure.

Example 20: VHH Monomers Protect CT26 Cells from TcdA

Cells of murine colorectal cancer cell line CT26 were exposed to TcdA (2ng/ml) for 24 hours and to a VHH monomer specific to TcdA (A3H, SEQ IDNO 61; A11G, SEQ ID NO:63; AC1, SEQ ID NO: 62; AE1, SEQ ID NO: 64; AH3,SEQ ID NO: A1; or AA6, SEQ ID NO: 60). Controls cells were exposed toTcdA (no VHH monomer was administered). The percentage of cell roundingwas monitored using a phase contrast microscope. Control cellsadministered only TcdA showed extensive cell rounding and distorted cellmorphology associated with TcdA toxin exposure.

It was observed that each of the VHH monomers reduced the percentage ofaffected cells and protected the cells from TcdA exposure (FIG. 25). Inorder of greatest VHH monomer activity to the weakest VHH monomeractivity, the greatest activity was observed for AA6, followed AH3, AC1,A3H, AE1, and A116 respectively. It was observed that VHH monomers AA6and AH3 neutralized TcdA and protected 50% of cells from toxincytotoxicity at VHH concentrations less than about 10 nM, and thus wereconsidered to have strong TcdA neutralizing activity.

Example 21: Multimeric Binding Proteins Protect Cells from TcdA

CT26 cells were contacted to TcdA (2 ng/ml) and concentrations (0.1 nM,0.48 nM, 2.4 nM, 12 nM, 60 nM, or 300 nM) of each of VHH monomers: A3H,A11G, AC1, AE1, AH3, or AA6. Control cells were administered toxin only.The strength of each neutralizing VHH activity was observed by analyzingprotection of cells from the toxin by VHH monomers. Percentage of cellrounding (% cell affected) caused by TcdA was monitored using a phasecontrast microscope (FIG. 25). Thus, the strongest VHH produced thegreatest protection at the lowest concentration. The VHHs wereidentified in the following order of efficacy: AA6 as the strongesttherapeutic agent, followed by AH3, AC1, AE1, A11G, and then A3H asweakest therapeutic agent.

To determine whether VHH monomers or VHH multimers effectivelyneutralized TcdA, CT26 cells were exposed for 24 hours to TcdA (2 ng/ml)and different concentrations (0.03 ng/mL, 0.1 ng/mL, 1 ng/mL, 3 ng/mL,10 ng/mL, 30 ng/mL, 100 ng/mL, 300 ng/mL, or 1000 ng/mL) of VHH monomers(AH3 or AA6), VHH heterodimer containing AH3 and AA6, or a homodimer ofthe heterodimer containing the heterodimer of AH3 and AA6 and fused toan artificial homodimerization domain called oAgB (Ah3/AA6/oAgB; SEQ IDNO: 95). The oAgB domain encodes a peptide having amino acid sequenceTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC (SEQ ID NO: 94) that bindsto proteins having the same sequence to form homodimers. The cysteine(amino acid abbreviation Cys or C) at the carboxyl end of AgBc becomesoxidized forming a covalent disulfide linkage between the two proteinmolecules to stabilize the homodimer (dimerizing sequence). Thus, theAH3/AA6 heterodimer itself becomes a homodimer containing two copies ofAH3/AA6 joined by the oAgBc dimerization domain (SEQ ID NO: 95). Controlcells were exposed to toxin only and not to VHH agents. The percentageof cell rounding (% cell affected) was monitored using a phase contrastmicroscope (FIG. 26).

Data show that control cells contacted with toxin only showed extensivetoxin mediated-cell rounding, and that each of the VHH monomers, AH3/AA6heterodimer and AH3/AA6/oAgB heterodimer/homodimer neutralized TcdA andprotected the CT26 cells from the toxin (FIG. 26). The AH3/AA6/oAgBheterodimer/homodimer displayed greatest activity to neutralize andprotect cells compared to the AH3/AA6 heterodimer, AH3 monomer, and AA6monomer respectively. The AH3/AA6/oAgB heterodimer/homodimer displayedabout three-fold stronger neutralizing activity for TcdA and protectionof the cells than the AH3/AA6 heterodimer alone, and about ten-foldbetter activity and protection than the VHH monomers (AH3 and AA6respectively).

Example 22: Heterodimer Binding Proteins Protect Cells from TcdA andTcdB

To determine activity of VHH heterodimers to neutralize both TcdA andTcdB, CT26 cells were exposed overnight to TcdA (2 ng/ml) or TcdB (0.1ng/ml), and then treated with a heterodimer composition containing VHH5D and VHH AA6 (FIG. 27 top graph) or with a heterodimer compositioncontaining VHH 5D and VHH AH3 (FIG. 27 bottom graph). Each heterodimerwas engineered to contain a VHH (5D) that strongly neutralized TcdB(FIG. 13) and to contain also a VHH (AA6 or AH3) that stronglyneutralized TcdA (FIG. 25). The percentage of cell rounding (% cellaffected) was monitored using a phase contrast microscope (FIG. 27 topand bottom graphs).

Data show that each of the 5D/AA6 heterodimer and the 5D/AH3 heterodimerneutralized both TcdA and TcdB (FIG. 27 top and bottom graphs). It wasobserved that 5D/AA6 heterodimer was about five-fold more effective inneutralizing TcdA than the 5D/AH3 heterodimer. Thus, the relativeneutralization strength of each heterodimer (FIG. 27) corresponded tothe relative neutralization strength of each corresponding AA6 monomerand AH3 monomer shown in FIGS. 25-26.

It was observed that the 5D/AA6 heterodimer was about three-fold orfour-fold more effective to neutralize TcdB than the 5D/AH3 heterodimer.Using a concentration of about 0.2 nM of administered 5D/AA6heterodimer, 50% of cells were protected, compared to about 1 nM of5D/AH3 heterodimer required for this same level of protection. Withoutbeing limited by any particular theory or mechanism of action, it ishere envisioned that the relative greater TcdA neutralization ability ofthe AA6 binding region compared to AH3 binding region resulted in asynergistically greater ability of the respective heterodimer toneutralize a separate toxin TcdB. The increased toxin neutralization for5D/AA6 for TcdB is presumably caused by amino acid sequences in TcdA andTcdB that are similar and are neutralized effectively by the AA6component of the heterodimer compared to the AH3 component of theheterodimer.

Example 23: 5D/AA6 Heterodimer Protected Subjects from C. difficileInfection

To further determine whether a single heterodimer could neutralize bothTcdA and TcdB and protect mice from oral C. difficile spore challenge, aprotocol for a clinically relevant mouse C. difficile infection model(Chen et al. 2008 Gastroenterology 135: 1984-1992) was performed asshown in FIG. 28. Groups of mice (ten mice/group) were treated to obtaina model of C. difficile associated diarrhea by treatment for three dayswith antibiotics in drinking water of the subjects, and then two dayslater by intraperitoneal administration of a single dose clindamycinbefore challenge with spores of a C. difficile strain expressing bothTcdA and TcdB (10⁶ spores/subject) on day zero (FIG. 28 A). To inducemore severe and fulminant disease, steroid dexamethasone was supplied tothe subjects in drinking water on day −6 (100 mg/mL) until day zero (Sunet al. 2001 Infection and Immunity 79: 2556-2864). Subjects wereintraperitoneally injected with VHH heterodimer containing 5D and AA6 (1mg/kg) three times (six hours, 16 hours, and 24 hours followinginoculation/challenge). Control subjects were similarly treated byinjection with PBS instead of the VHH heterodimer. Subjects weremonitored hours and days following the VHH injection.

Data show that 100% of control subjects administered toxin died withintwo days of toxin challenge (FIG. 28 B) and suffered diarrhea (FIG. 28C). Only 20% of subjects administered 5D/AA6 heterodimer developeddiarrhea and 90% survived (FIGS. 28 B and C). Thus, 5D/AA6 heterodimerprotected subjects from both TcdA and TcdB spore challenge in aclinically relevant mouse C. difficile infection model.

Example 24: Recombinant Multimeric Binding Proteins Neutralize aPlurality of Disease Agents

Effectiveness of the antitoxin treatment using multimeric bindingproteins are analyzed by determine ability of the binding proteins tobind to and neutralize a disease agent target.

Recombinant heteromultimeric neutralizing binding protein containingmultiple binding regions with or without epitopic tags are produced. Thebinding regions are not identical and each binding region has affinityto specifically bind a non-overlapping portion of a disease agent: TcdAtoxin, TcdB toxin, and a Shiga toxin. The genes encoding proteins aremultimerized to form different heteromultimeric binding proteins usingthe oAgBc dimerization domain (SEQ ID NO: 94) shown in Example 21.

Subjects are exposed to a mixture of disease agents (TcdA toxin, TcdBtoxin, Shiga toxin and a norovirus), and then are administered each ofthe heteromultimeric binding proteins, or a mixture of monoclonalantibodies specific for either TcdA, TcdB, Shiga Toxin 1, and thenorovirus. Control subjects are administered the mixture of diseaseagents only (no multimeric binding proteins). Subjects are monitored forindicia of exposure to the pathogenic molecules such as diarrhea, fever,tachycardia, respiratory distress, and death.

Meyer-Kaplan plots quantifying survival of subjects are prepared andweeks later remaining subjects are sacrificed to analyze tissue and cellmorphology. A surprising synergistic protective effect is observed forsubjects administered the multimeric binding proteins with or withoutepitopic tags. Data show that subjects administered the multimericbinding proteins survive longer and have little or no indicia ofexposure to the mixture of disease agents compared results for subjectsadministered monoclonal antibodies to each disease agent and for controlsubjects administered only disease agents. Subject administeredheteromultimeric binding proteins specific for disease agents do notexperience diarrhea, fever or other indicia of exposure to the diseaseagents. Tissues from subjects administered multimeric binding proteinsshow normal cell appearance without signs of cell rounding or cell lysiscaused by either TcdA, TcdB, Shiga Toxin 1, and the norovirus. Themultimeric binding proteins neutralize each of these disease agents.Control subjects have diarrhea, and tissues excised from the intestinalsystems show indicia of colitis and extensive internal bleeding.

The multimeric binding protein specific for a mixture of bacterialtoxins and a viral infectious agent neutralize each of the diseaseagents and protected the cells from the subjects from cytotoxicity andcell lysis.

Example 25: Materials and Methods

Purified, catalytically inactive mutant forms of full-length recombinantdisease agent (shiga toxin, anthrax protective antigen, ricin A chaintoxin, or ricin B chain) were obtained as described in Tremblay et al.pages 4593-4594. Shiga toxins were obtained from Phoenix Lab at TuftsMedical Center. Purified anti-Stx1 monoclonal antibody (mAb) 4D3,anti-Stx2 mAbs 3D1 and 5C12, and recombinant Stx1 B chain andrecombinant Stx2 A and B chain were kindly provided by Dr. AbhineetSheoran. Stx1 and Stx2 toxoids were prepared by formalin inactivation ofthe holotoxins and then dialyzed. Reagents for Western blotting werepurchased from KPL Inc. (Gaithersburg, Md.). Antibodies used wereanti-E-tag mAb (Phadia; Uppsala, Sweden); HRP-anti-E-tag mAb (BethylLaboratories Inc.; Montgomery, Tex.); HRP-anti-M13 Ab (GE Healthcare;Woburn, Mass.). Tremblay et al. pages 4593-4594 describe in detail thematerials and methods used in examples herein.

Example 26: VHHs that Bind and Shiga Toxin

VHH binding agents were produced, purified and were screened to identifythose that specifically bind to Shiga toxins. It was observed that oneresulting VHHH, JET-H12, bound specifically to both Shiga-like toxin(Stx) 1 and Stx2. Another VHH, JFG-H6, was observed to bind specificallyto Stx2 (See FIG. 29 A-B). The amino acid sequences and nucleotidesequences for JET-H12 and JFG-H6 were determined and are shown below:

JET-H12 (SEQ ID NO: 96)QVQLVETGGGLVQAGDPLRLSCVASGRTVSRYDKAWFRQAPGKEREFVAGISWNGDTKIYADSVKGRFTISRENSRDTLDLQIDNLKPEDTAAYYCAVGIAGVQSMARMLGVRYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 97)CAGGTGCAGCTCGTGGAGACGGGGGGAGGATTGGTGCAGGCTGGGGACCCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTCGCTATGACAAGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGAATTAGCTGGAACGGCGATACAAAAATTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAACTCCAGGGATACACTGGATCTGCAAATTGACAACCTGAAACCTGAGGACACGGCCGCGTATTACTGTGCGGTCGGAATTGCGGGTGTTCAGAGTATGGCGCGTATGCTCGGAGTGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JFG-H6(SEQ ID NO: 98) QVQLVETGGGLVQPGGSLRLSCAASGFSLDPYVIGWFRQAPGKEREGVSCITSRAASRTSVDSVNERFTISRDNAKNTVDLHINNLKPEDSGVYYCAAVPPAKLPLFSLCRSLPAKYDYWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 99)CAGGTGCAGCTCGTGGAGACGGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGTTTCAGTTTGGACCCTTATGTGATAGGATGGTTCCGGCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTACGAGTAGGGCTGCTAGTCGAACGTCTGTAGACTCCGTGAACGAGCGATTCACCATCTCCAGAGACAACGCCAAGAATACGGTCGATCTACACATCAATAACCTGAAACCTGAGGACTCGGGCGTTTATTACTGTGCAGCGGTCCCCCCTGCCAAATTACCACTTTTCAGCCTATGTCGCTCCCTGCCAGCAAAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACA GCGAAGACCCCTCG;

Example 27: VHHs that Bind Anthrax Protective Antigen

VHH binding agents were produced, purified and identified that arespecific to anthrax protective antigen (PA) positive VHHs (See FIG. 29A-B). It was observed that the following VHHs specifically bind anthraxPA: JHD-B6, JHE-D9, JIJ-A12, JIJ-B8, JIJ-C11, JIJ-D3, JIJ-F11, JIK-B8,JIK-B1, JIK-B12, and JIK-F4. The amino acid sequence and nucleotidesequence of each of these VHHs were determined and are shown below:

JHD-B6 (SEQ ID NO: 100)QVQLVESGGGLVQPGGSLRLSCAASGSSFSRYAMRWYRQAPGKQRELVANINSRGTSNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAEWLGRSEPSWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 101)CAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGTAGCTTCAGTAGATATGCCATGCGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAAACATTAATAGTCGTGGTACCTCAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGCCGTCTATTATTGTAATGCAGAGTGGTTGGGACGATCGGAGCCTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAA; JHE-D9 (SEQ ID NO: 102)QVQLVESGGGLVQPGGSLRLSCAASGFIFSLYTMRWHRQAPGKERELVATITSATGITNYADSVKGRFIISRDDAKKTGYLQMNSLKPEDTAVYYCNAVRTTVSRDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 103)CAGGTGCAGCTCGTGGAGTCAGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTCTTTATACCATGAGGTGGCACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCGACTATTACTAGTGCTACTGGTATTACAAACTATGCAGACTCCGTGAAGGGCCGATTCATCATCTCCAGAGACGATGCCAAGAAGACGGGGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTAATGCAGTCCGCACTACCGTGTCACGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIJ-A12 (SEQ ID NO: 104)QVQLVESGGGLVQPGGSLRLSCAASGIIFSIYTMGWYRQAPGKQRELVAAIPSGPSANATDSVGGRFTITRDNAENTVYLQMNDLKPEDTAVYYCNARRGPGIKNYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 105)CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATCTTCAGTATCTATACCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCAGCTATACCTAGTGGTCCTAGCGCAAACGCTACAGACTCCGTGGGGGGCCGATTCACCATCACCAGAGACAACGCCGAGAACACGGTGTATCTGCAAATGAACGACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCTCGGCGGGGTCCGGGTATCAAAAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIJ-B8 (SEQ ID NO: 106)QVQLVESGGGLVQPGGSLSVSCAASGSIARPGAMAWYRQAPGKERELVASITPGGLTNYADSVTGRFTISRDNAKRTVYLQMNSLQPEDTAVYYCHARIIPLGLGSEYRDHWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 107)CAGGTGCAGCTCGTGGAGTCCGGGGGCGGCTTGGTGCAGCCCGGGGGGTCTCTGAGTGTCTCCTGTGCAGCCTCTGGAAGCATCGCAAGACCAGGTGCCATGGCCTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCGTCTATTACGCCTGGTGGTCTTACAAACTATGCGGACTCCGTGACGGGCCGATTCACCATTTCCAGAGACAACGCCAAGAGGACGGTGTATCTGCAGATGAACAGCCTCCAACCCGAGGACACGGCCGTCTATTACTGTCATGCACGAATAATTCCCCTAGGACTTGGGTCCGAATACAGGGACCACTGGGGCCAGGGGACTCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCTCG; JIJ-C11 (SEQ ID NO: 108)QVQLVETGGGLVQPGGSLGLSCVVASGRSINNYGMGWYRQAPGKQRELVAQISSGGTTNYAGSVEGRFTISRDNVKKMVYLQMNSLKPEDTAVYYCNSLLRTFSWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 109)CAGGTGCAGCTCGTGGAGACGGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGGGACTCTCCTGTGTAGTCGCCTCTGGAAGAAGCATCAATAATTATGGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGCAAATTAGTAGTGGTGGTACCACAAATTATGCAGGCTCCGTAGAGGGCCGATTCACCATCTCCAGAGACAACGTCAAGAAAATGGTGTATCTTCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATTCACTGCTCCGAACTTTTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGCA CCACAGCGAAGACCCCTCG;JIJ-D3 (SEQ ID NO: 110)QVQLVETGGLVQPGGSLRLSCAASGLTFSSTAMAWFRQAPGKEREFVARISGAGITIYYSDSVKDRFTISRNNVENTVYLQMNSLKTEDTAVYYCAARRNTYTSDYNIPARYPYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 111)CAGGTGCAGCTCGTGGAGACCGGGGGGTTGGTGCAGCCTGGGGGCTCCCTGCGACTCTCCTGTGCAGCCTCCGGACTCACCTTCAGTAGCACTGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGTATTAGCGGGGCTGGTATTACGATCTACTATTCGGACTCCGTGAAGGACCGATTCACCATCTCCAGAAACAACGTCGAGAACACGGTGTATTTGCAAATGAACAGCCTGAAAACTGAGGACACGGCCGTTTACTACTGTGCAGCAAGACGGAATACTTACACTAGCGACTATAACATACCCGCCCGGTATCCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIJ-E9(SEQ ID NO: 112) QVQLVETGGLVQPGGSLRLSCAASRSTTATIYSMNWYRQAPGKQRELVAGMTSDGQTNYATSVKGRFTISRDNAKNTVYLLMNSLKLEDTAVYYCYVKPWRLQGWDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 113)CAGGTGCAGCTCGTGGAGACGGGGGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGCACGACGGCCACAATTTATAGTATGAACTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGGGTATGACTAGTGATGGTCAGACAAACTATGCAACCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTATATTTGCTAATGAACAGCCTGAAACTTGAGGACACGGCCGTCTATTATTGTTATGTAAAACCATGGAGACTACAAGGTTGGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIJ-F11 (SEQ ID NO: 114)QVQLVESGGGLVQPGGSLRLSCAAPESIVNSRTMAWYRQAPGKQRERVATITTAGSPNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNTLLSTLPYGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 115)CAGGTGCAGCTCGTGGAGTCGGGCGGCGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCCCTGAAAGCATCGTCAATAGCAGAACCATGGCCTGGTACCGCCAGGCTCCAGGAAAGCAGCGCGAAAGGGTCGCCACTATTACTACTGCTGGTAGCCCAAATTATGCAGACTCTGTGAAGGGCCGATTCGCCATCTCCAGAGACAACGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGCAATACACTTCTCAGCACCCTTCCCTATGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGCACCA CAGCGAAGACCCCTCG;JIK-B8 (SEQ ID NO: 116)QVQLVESGGGLVQPGGSLGLSCVVASERSINNYGMGWYRQAPGKQRELVAQISSGGTTNYADSVEGRFTISRDNVKKMVHLQVNSLKPEDTAVYYCNSLLRTFSWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 117)CAGGTGCAGCTCGTGGAGTCGGGCGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGGGACTCTCCTGTGTAGTCGCCTCTGAAAGAAGCATCAATAATTATGGCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGCAAATTAGTAGTGGTGGTACCACAAATTATGCAGACTCCGTAGAGGGCCGATTCACCATCTCCAGAGACAACGTCAAGAAAATGGTGCATCTTCAAGTGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATTCGCTACTCCGAACTTTTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACC CAAGACACCAAAACCACAA;JIK-B10 (SEQ ID NO: 118)QVQLVETGGGLVQPGGSLRLSCAASGFTFSSYRMSWYRQAAGKERDVVATITANGVPTGYADSVMGRFTISRDNAKNTVYLEMNSLNPEDTAVYYCNAPRLHTSVGYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 119)CAGGTGCAGCTCGTGGAGACGGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATCGCATGAGCTGGTACCGGCAGGCTGCAGGGAAGGAGCGCGACGTGGTCGCAACAATTACTGCTAATGGTGTTCCCACAGGCTATGCAGACTCCGTGATGGGCCGATTCACCATTTCCAGAGACAATGCCAAGAACACGGTGTATCTGGAAATGAACAGCCTGAATCCTGAGGACACGGCCGTGTATTACTGTAACGCGCCCCGTTTGCATACATCTGTAGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIK-B12 (SEQ ID NO: 120)QVQLVESGGGLVQAGNSLRLSCTASGVIFSIYTMGWFRQAPGKEREFVAAIGVADGTALVADSVTGRFTISRDNAKNTVYLHMNSLKPEDTAVYSCAAYLSPRVQSPYITDSRYQLWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 121)CAGGTGCAGCTCGTGGAGTCGGGAGGAGGATTGGTGCAGGCTGGGAACTCTCTGAGACTCTCCTGTACGGCCTCTGGTGTGATCTTCTCTATCTATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCGATAGGGGTGGCTGATGGTACCGCACTTGTGGCAGACTCCGTGACGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACCGTTTATCTGCATATGAACAGCCTGAAGCCTGAGGACACGGCCGTCTATTCCTGTGCAGCGTATCTTAGCCCCCGTGTCCAATCCCCCTACATAACTGACTCCCGGTATCAACTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAAC CACAA JIK-F4(SEQ ID NO: 122) TGGGLVQAGGSLRLSCAASGRYAMGWFRQAPGKEREFVATISRSGAIREYADSVKGRFTISRDGAENTVYLEMNSLKPDDTAIYVCAEGRGATFNPEYAY WGQGTQVTVSSAHHSEDPS;(SEQ ID NO: 123) CAGGTGCAGCTCGTGGAGACTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGGCTCTCCTGTGCAGCCTCTGGACGCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCGACTATTAGCCGGAGTGGTGCTATCAGAGAGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGGCGCCGAGAACACGGTGTATCTGGAAATGAACAGCCTGAAACCTGACGACACGGCCATTTATGTCTGTGCAGAAGGACGAGGGGCGACATTCAACCCCGAGTATGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCTCG;

Example 28: VHHs that Bind to Ricin Toxin A

VHH binding agents were produced, purified and identified that arespecific to ricin toxin A chain (RTA; see FIG. 29 A-B). The followingVHHs were determined to specifically bind RTA: JIV-F5, JIV-F6, JIV-G12,JIY-A7, JIY-D9, JIY-D10, JIY-E1, JIY-E3, JIY-E5, JIY-F10 and JIY-G11.The amino acid sequence and nucleotide sequence of each of these VHHswere determined and are shown below:

JIV-F5 (SEQ ID NO: 124)QVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQVPGKEREGVACVKDGSTYYADSVKGRFTISRDNGAVYLQMNSLKPEDTAVYYCASRPCFLGVPLIDFGSWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 125)CAGGTGCAGCTCGTGGAGTCGGGCGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATGATTATGCCATAGGCTGGTTCCGCCAGGTCCCAGGGAAGGAGCGTGAGGGGGTCGCATGTGTTAAAGATGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGGCGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCATCCAGGCCCTGCTTTTTGGGTGTACCACTTATTGACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAA; JIV-F6 (SEQ ID NO: 126)QVQLVESGGGLVQAGGSLRLSCATSGGTFSDYGMGWFRQAPGKEREFVAAIRRNGNGGNGIEYADSVKGRFTISRDNAKNTVHLQMNSLTPEDTAVYYCAASISGYAYNTIERYNYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 127)CAGGTGCAGCTCGTGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGCGCAACCTCTGGCGGCACCTTCAGTGACTATGGAATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGGCGGAATGGTAATGGCGGTAATGGCATTGAATATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGCATCTACAAATGAACAGCCTGACACCTGAGGACACGGCCGTTTATTACTGTGCAGCGTCAATATCGGGATACGCTTATAACACAATTGAAAGATATAACTACTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCAGGAACCCAAGACACCAAAA CCACAA; JIV-G12(SEQ ID NO: 128) QVQLVESGGGLVQAGGSLSLSCAASGGDFSRNAMAWFRQAPGKEREFVASINWTGSGTYYLDSVKGRFTISRDNAKNALYLQMNNLKPEDTAVYYCARSTVFAEITGLAGYQSGSYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 129)CAGGTGCAGCTCGTGGAGTCCGGCGGAGGATTGGTGCAGGCGGGGGGCTCTCTGAGTCTCTCCTGTGCAGCCTCTGGAGGTGACTTCAGTAGGAATGCCATGGCCTGGTTCCGTCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCATCTATTAACTGGACTGGTAGTGGCACATATTATCTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCCCTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCACGCTCCACGGTGTTTGCCGAAATTACAGGCTTAGCAGGCTACCAGTCGGGATCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACAC CAAAACCACAA; JIY-A7(SEQ ID NO: 130) QVQLVETGGGTVQTGGSLRLSCSASGGSFSRNAMGWFRQAPGKEREFVAAINWSASSTYYRDSVKGRFTVSRDNAKNTVYLHLNSLKLEDTAAYYCAGSSVYAEMPYADSVKATSYNYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 131)CAGGTGCAGCTCGTGGAGACCGGCGGAGGAACGGTGCANACTGGGGGCTCTCTGAGACTCTCCTGTTCAGCCTCTGGCGGCTCCTTCAGTAGGAATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCTATTAACTGGAGTGCCTCTAGTACTTATTATAGAGACTCCGTGAAGGGACGATTCACCGTCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCATTTGAACAGCCTGAAACTTGAGGACACGGCCGCGTATTACTGTGCTGGAAGCTCGGTGTATGCAGAAATGCCGTACGCCGACTCTGTCAAGGCAACTTCCTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACAC CAAAACCACAA; JIY-D9(SEQ ID NO: 132) QVQLVETGGGLVQAGGSLRLPCSFSGFPFDNYFVGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGADFLTPHRCPALYDYWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 133)CAGGTGCAGCTCGTGGAGACCGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCCCCTGTTCATTCTCTGGATTCCCTTTCGATAATTATTTCGTAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGTCTGAAACCTGAGGATACGGCCGTTTATTACTGTGGAGCAGATTTCCTCACCCCACATAGGTGTCCAGCCTTATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCTCG; JIY-D10 (SEQ ID NO: 134)QVQLVESGGGLVQPGGSLRLHCAASGSIASIYRTCWYRQGTGKQRELVAAITSGGNTYYADSVKGRFTISRDNAKNTIDLQMNSLKPEDTAVYYCNADEAGIGGFNDYWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 135)CAGGTGCAGCTCGTGGAGTCTGGTGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCCACTGTGCAGCCTCTGGAAGCATCGCCAGTATCTATCGCACGTGCTGGTACCGCCAGGGCACAGGGAAGCAGCGCGAGTTGGTCGCAGCCATTACTAGTGGTGGTAACACATACTATGCGGACTCCGTTAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACACAATCGATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGACGAGGCGGGGATCGGGGGATTTAATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCTCG; JIY-E1 (SEQ ID NO: 136)QVQLVESGGGLVQAGGSLRLSCAASGRTFSRSSMGWFRQAPGKEREFVASIVWADGTTLYGDSVKGRFTVSRDNVKNMVYLQMNNLKPEDTALYYCADNKFVRGLVAVRAIDYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 137)CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTCGCAGTTCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTCGTTGCGTCCATTGTCTGGGCTGATGGTACGACGTTGTATGGAGACTCCGTAAAGGGCCGATTCACCGTCTCCAGGGACAACGTCAAGAACATGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCCTTTATTACTGTGCGGACAATAAATTCGTCCGTGGATTAGTGGCTGTCCGTGCGATAGATTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCGTCAGAACCCAAGACACCAAAACCAC AA; JIY-E3(SEQ ID NO: 138) QVQLVESGGLVQAGGSLRLSCAASGRADIIYAMGWFRQAPGKEREFVAAVDWSGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAARRSWYRDALSPSRVYEYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 139)CAGGTGCAGCTCGTGGAGTCGGGAGGATTGGTGCAGGCTGGAGGCTCTCTGAGACTCTCCTGCGCAGCCTCTGGACGCGCCGACATAATCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCGGCAGTAGACTGGAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCCGAAGGAGCTGGTACCGAGACGCGCTATCCCCCTCCCGGGTGTATGAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAAC CACAA; JIY-E5(SEQ ID NO: 140) QVQLVETGGGLVQPGGSLTLSCAGSGGTLEHYAIGWFRQAPGKEHEWLVCNRGEYGSTVYVDSVKGRFTASRDNAKNTVYLQLNSLKPDDTGIYYCVSGCYSWRGPWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 141)CAGGTGCAGCTCGTGGAGACGGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCAGGCTCCGGTGGCACTTTGGAACATTATGCTATAGGCTGGTTCCGCCAGGCCCCTGGGAAAGAGCATGAGTGGCTCGTATGTAATAGAGGTGAATATGGGAGCACTGTCTATGTAGACTCCGTGAAGGGCCGATTCACCGCCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAATTGAACAGTCTGAAACCTGACGACACAGGCATTTATTACTGTGTATCGGGATGTTACTCCTGGCGGGGTCCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGCACCACAGCGAAGACCCCTCG; JIY-F10 (SEQ ID NO: 142)QVQLVESGGGLVQPGGSLKLSCRASGSIVSIYAVGWYRQAPGKQRELLAAITTDGSTKYSDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAIYSCIGDAAGWGDQYYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 143)CAGGTGCAGCTCGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCTGTAGAGCCTCTGGAAGCATAGTCAGTATCTATGCCGTGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGCTCGCGGCTATCACTACTGATGGTAGCACGAAGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACGGTATATCTGCAAATGAACAACCTCAAACCTGAGGACACGGCCATCTATTCCTGTATCGGGGACGCGGCGGGTTGGGGCGACCAATACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIY-G11 (SEQ ID NO: 144)QVQLVESGGGLVQAGGSLRLSCAASGSIVNFETMGWYRQAPGKERELVATITNEGSSNYADSVKGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCSATFGSRWPYAHSDHWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 145)CAGGTGCAGCTCGTGGAGTCAGGCGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCGTCAATTTCGAAACCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCAACTATTACTAATGAAGGTAGTTCAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACACGGTGTCCCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTACTACTGTTCGGCGACGTTCGGCAGTAGGTGGCCGTACGCCCACAGTGATCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA;

Example 29: VHHs Specific for Ricin Toxin B

VHH binding agents were produced, purified and identified that arespecific to ricin toxin B chain (See FIG. 29 A-B). VHHs thatspecifically bind RTB were determined and are: JIW-B1, JIW-C12, JIW-D12,JIW-G5, JIW-G10, JIZ-B7, JIZ-B9, JIZ-D8, and JIZ-G4. The amino acidsequence and nucleotide sequences of each of these VHHs were determinedand are shown below:

JIW-B1 (SEQ ID NO: 146)QVQLVETGGALVHTGGSLRLSCEVSGSTFSSYGMAWYRQAPGEQRKWVAGIMPDGTPSYVNSVKGRFTISRDNAKNSVYLHMNNLRPEDTAVYYCNQWPRTMPDANWGRGTQVTVSSEPKTPKPQ; (SEQ ID NO: 147)CAGGTGCAGCTCGTGGAGACGGGCGGAGCATTGGTGCACACTGGGGGTTCTCTGAGACTCTCCTGCGAAGTCTCCGGAAGCACCTTCAGTAGCTATGGCATGGCCTGGTACCGCCAAGCTCCAGGCGAGCAGCGTAAGTGGGTCGCAGGTATTATGCCGGATGGTACTCCAAGCTATGTAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTGTATCTGCACATGAACAACCTGAGGCCTGAAGACACGGCCGTCTATTATTGCAACCAATGGCCGCGCACGATGCCTGACGCGAACTGGGGCCGGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIW-C12 (SEQ ID NO: 148)QVQLVETGGSLRLTCVTSGSTFNNPAITWYRQPPGKQREWVASLRSGDGPVYRESVKGRFTIFRDNATDALYLRMNSLKPEDTAVYHCNTASPASWLDWG QGTQVTVSSEPKTPKPQ;(SEQ ID NO: 149) CAGGTGCAGCTCGTGGAGACTGGGGGGTCTCTGAGGCTCACCTGTGTAACCTCTGGAAGCACCTTCAATAATCCTGCCATAACCTGGTACCGCCAGCCTCCAGGGAAGCAGCGTGAGTGGGTCGCAAGTCTTCGTAGTGGTGATGGTCCAGTATATAGGGAATCCGTGAAGGGCCGATTCACCATTTTTAGAGACAACGCCACGGACGCGCTGTATCTGCGGATGAATAGCCTGAAACCTGAGGACACGGCCGTCTATCACTGTAACACCGCCTCACCTGCTAGTTGGCTGGACTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCAGAACCCAAGACACCAAAACCACA A; JIW-D12(SEQ ID NO: 150) QVQLVETGGGLVQPGGSLRLSCATSGFPFSTERMSWVRQAPGKGLEWVSGITEGGETTLAAPSVKGRFNISRDNARNILYLQMNSLKPEDAAVYYCFRGVFFRTSFPPELARGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 151)CAGGTGCAGCTCGTGGAGACGGGAGGAGGATTGGTGCAACCTGGGGGTTCTCTGAGACTCTCTTGTGCAACCTCTGGATTCCCCTTCAGTACGGAGCGTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTACTGAGGGTGGTGAAACCACTCTCGCGGCACCCTCCGTGAAGGGCCGATTCAACATCTCCAGAGACAACGCCAGGAATATCCTATATCTACAGATGAATTCCTTGAAACCTGAGGACGCGGCCGTTTACTATTGTTTTAGAGGTGTTTTTTTTAGAACGAGTTTTCCTCCCGAACTCGCGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIW-G5 (SEQ ID NO: 152)QVQLVESGGGLVQAGGSLRLSCAASGSAVSDSFSTYAISWHRQAPGKQREWIAGISNRGATSYRDSVKGRFTISRDNAKNTVYLQMNNLKPEDTGVYYCEPWPREGLGGGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 153)CAGGTGCAGCTCGTGGAGTCGGGCGGAGGCTTGGTGCAGGCAGGGGGGTCTTTGAGACTCTCCTGTGCAGCCTCTGGAAGCGCCGTCAGTGACAGCTTCAGTACCTATGCCATCTCCTGGCACCGCCAGGCTCCAGGGAAGCAGCGTGAGTGGATCGCAGGTATTAGTAATCGTGGTGCGACAAGCTATAGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTATATCTGCAAATGAACAACCTGAAACCTGAGGACACGGGCGTCTATTATTGTGAGCCATGGCCACGCGAAGGACTTGGGGGGGGCCAGGGGACTCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA; JIW-G10 (SEQ ID NO: 154)QVQLVESGGGSVQTGGSLTLSCVVSGSTFSDYAVAWYRQVPGKSRAWVAGVSTTGSTSYTDSVRGRFTISRDNHKKTVYLSMNSLKPEDTGIYYCNLWPFTNPPSWGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 155)CAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTCGGTGCANACTGGGGGGTCTCTGACACTCTCCTGTGTAGTCTCTGGAAGTACCTTCAGTGACTATGCGGTGGCCTGGTACCGCCAGGTTCCAGGCAAATCGCGTGCGTGGGTCGCGGGTGTTAGTACTACTGGCTCGACATCTTATACAGACTCCGTGAGGGGCCGGTTCACCATCTCCAGAGACAACCACAAGAAGACGGTGTATCTTTCAATGAACAGCCTGAAACCTGAGGACACGGGCATCTATTACTGCAACTTATGGCCGTTCACAAATCCTCCTTCCTGGGGCCAGGGAACCCAAGTCACCGTTTCCTCGGCGCACCACAGCGAAGACCCCTCG; JIZ-B7 (SEQ ID NO: 156)QVQLVESGGAVVQPGGSLRLSCATSGFTFSDDRMSWARQAPGKGLEWVSGISTASEGFATLYAPSVKGRFTISRDNAKHMLYLQMDTLKPEDTAVYYCLRGVFFRTNIPPEVLRGQGTQVTVSSAHHSEDPS; (SEQ ID NO: 157)CAGGTGCAGCTCGTGGAGTCTGGAGGAGCCGTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTCAGTGACGATCGTATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTAGTACTGCTAGTGAAGGTTTTGCTACACTCTACGCACCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGCATATGCTGTATCTGCAAATGGATACCTTGAAACCTGAGGACACGGCCGTGTATTACTGTTTAAGAGGGGTTTTTTTTAGAACGAACATTCCTCCCGAGGTACTGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCCTCG; JIZ-B9 (SEQ ID NO: 158)QVQLVETGGDLVQPGGSLRLSCAASGSSFSRAAVGWYRQAPGKEREWVARLASGDMTDYTESVRGRFTISRDNAKHTVYLQMDNLKPEDTAVYYCKARIPPYYSIEYWGKGTRVTVSSEPKTPKPQ; (SEQ ID NO: 159)CAGGTGCAGCTCGTGGAGACGGGGGGAGACTTGGTGCANCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCTTCAGCCGCGCTGCCGTGGGCTGGTACCGTCAGGCTCCAGGAAAGGAGCGTGAGTGGGTCGCACGTCTCGCGAGTGGTGATATGACGGACTATACCGAGTCCGTGAGGGGCCGATTCACTATCTCCAGAGACAACGCCAAGCACACGGTGTATCTGCAAATGGACAACCTGAAACCTGAGGACACGGCCGTCTACTATTGTAAGGCCAGGATACCCCCTTATTACTCTATAGAGTACTGGGGCAAAGGGACCCGGGTCACCGTCTCCTCANAACCCAAGACACCAAAACCACAA; JIZ-D8 (SEQ ID NO: 160)QVQLVETGGGLVQAGGSLRLSCVVSSPLFNLYDMAWYRQAPGNQRELVAGILTDGRATYSDSVKGRFTISRNNLTNTVFLQMSSLKPEDTAVYYCNRKNSIYWDSWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 161)CAGGTGCAGCTCGTGGAGACAGGTGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAGTATCTAGTCCCCTGTTCAATCTTTACGACATGGCCTGGTATCGCCAGGCTCCAGGGAATCAGCGTGAGTTGGTCGCAGGCATCTTGACTGATGGTCGCGCAACATATTCAGACAGCGTGAAGGGCCGATTCACCATTTCCAGAAACAACCTGACGAACACGGTGTTTTTACAAATGAGCAGCCTGAAACCTGAGGACACGGCCGTCTATTATTGTAATAGAAAGAATAGTATCTACTGGGATTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGAACCCAAGACACCAAAACCACAA; JIZ-G4 (SEQ ID NO: 162)QVQLVESGGGLVQAGGSLRLSCVASGLTFSRYGMGWFRQAPGQERVVVSVISPDGGSAYYADSVKGRFTISRDNAKNTVYLQMSTLRFEDTGVYYCTAGPRNGATTVLRPGDYDYWGQGTQVTVSSEPKTPKPQ; and (SEQ ID NO: 163)CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGCGTAGCCTCTGGACTCACCTTCAGTCGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGACAGGAGCGTGTAGTCGTATCAGTTATTAGTCCCGACGGTGGTAGCGCATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAGCACCCTGAGATTTGAGGACACGGGCGTTTATTATTGTACAGCAGGGCCCCGGAATGGAGCGACTACAGTCCTCCGGCCAGGGGATTATGACTACTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCAGAACCCAAGACACCAAAACCAC AA

Example 30: VHH Binding Proteins Bind to Neutralized Toxin-DiseaseAgents

Effectiveness of the antitoxin treatment using VHH binding proteinscomposed of SEQ ID NOs:96-163 were analyzed to determine ability of thebinding proteins to bind to and neutralize a toxin disease agent target(see Tremblay et al. and FIGS. 29 A and B). Data show that the VHHeffectively bound to and neutralized Stx 1, Stx2, anthrax toxins, RTA,and RTB.

Example 31: Recombinant Multimeric Binding Proteins Neutralize aPlurality of Disease Agents

Recombinant heteromultimeric neutralizing binding proteins containingmultiple binding regions composed of any of SEQ ID NOs: 96-163 areproduced. At least two of the binding regions are not identical and eachbinding region has affinity to specifically bind a non-overlappingportion of a disease agent associated with toxin proteins produced bybacteria or plants such as a Shiga toxin, a ricin toxin (e.g., RTA andRTB), and anthrax toxin.

Subjects are exposed to one or more of Shiga toxin, ricin toxin A chain,and ricin toxin B chain, and then are administered each of theheteromultimeric binding proteins. Control subjects are administered theone or more disease agents only (no multimeric binding proteins).Subjects are monitored for indicia of exposure to the pathogenicmolecules such as diarrhea, fever, tachycardia, respiratory distress,and death.

Subject administered heteromultimeric binding proteins specific fordisease agents are observed to have little or no indicia of exposure tothe one or more disease agents. In vitro analysis of cell, blood andtissue samples from the subjects show that the multimeric bindingproteins neutralize each of these disease agents in the samples. Controlsubjects show indicia of being exposed to the disease agents (e.g.,diarrhea, internal bleeding, and cell lysis). Thus, the recombinantheteromultimeric neutralizing binding proteins are found to be effectiveinhibitors of the toxin disease agents.

Example 32: VHHs that Bind and Neutralize Plant Toxins

Methods as described in Examples herein using phage libraries are usedto produce and identify VHHs that specifically bind and neutralize planttoxins. The VHHs specifically neutralize each of the following planttoxins: Akar saga (Abrus precatorius), Deathcamas, Amianthium Angel'sTrumpet (Brugmansia), Angel Wings (Caladium), Anticlea, Autumn crocus(Colchicum autumnale), Azalea (Rhododendron), Bittersweet nightshade(Solanum dulcamara), Black hellebore (Helleborus niger), Black locust(Robinia pseudoacacia), Black nightshade (Solanum nigrum), Bleedingheart (Dicentra cucullaria), Blind-your-eye mangrove (Excoecariaagallocha), Blister Bush (Peucedanum galbanum), Bloodroot (Sanguinariacanadensis), Blue-green algae (Cyanobacteria), Bobbins (Arum maculatum),Bracken (Pteridium aquilinum), Broom (Cytisus scoparius), calabar bean(Physostigma venenosum), castor bean, Christmas rose (Helleborus niger),Columbine (Aquilegia), Corn cockle (Agrostemma githago), corn lily(veratrum), cowbane (Cicuta), cows and bulls (Arum maculatum), crab'seye (Abrus precatorius), cuckoo-pint (Arum maculatum), daffodil(Narcissus), Darnel (Lolium temulentum), Deadly nightshade (Atropabelladonna), Devils and angels (Arum maculatum), False acacia (Robiniapseudoacacia), False hellebore (Veratrum), Foxglove (Digitalispurpurea), Frangipani (Plumeria), Doll's eyes (Actaea pachypoda),Dumbcane (Dieffenbachia), Dutchman's breeches (Dicentra cucullaria),Elder/Elderberry (Sambucus), Giant hogweed (Heracleum mantegazzianum),Giddee giddee, Gifblaar (Dichapetalum cymosum), Greater celandine(Chelidonium majus), Gympie gympie (Dendrocnide moroides), Heart ofJesus (Caladium), hemlock (Conium maculatum), hemlock water-dropwort(Oenanthe crocata), henbane (Hyoscyamus niger), Horse chestnut (Aesculushippocastanum), Holly (Ilex aquifolium), Hyacinth (Hyacinthusorientalis), Indian licorice, Jack in the pulpit, Jamestown weed,jequirity, Jerusalem cherry, Jimson weed, John Crow bead, Jumbie bead,Lily of the Valley, Lords and Ladies, Madiera winter cherry, Mayapple,Meadow saffron, Milky mangrove, Monkshood, Moonseed, Passion flower,Plumeria, Poison hemlock, Poison ivy, Poison oak, Poison parsnip, Poisonsumac, Poison ryegrass, Pokeweed, Precatory bean, Privet, ragwort,redoul, River poison tree, Robinia pseudoacacia (also known as blacklocust and false acacia), Rosary pea, Sosnowsky's Hogweed, Spindle tree,Starch-root, Stenanthium, Stinging tree, Stinkweed, Strychnine tree,Suicide tree (Cerbera odollam), thorn apple, Toxicoscordion, Wake robin,Water hemlock, White baneberry, White snakeroot, Wild arum, Wintercherry, Wolfsbane, Yellow Jessamine, Yew, and Zigadenus.

Example 33: Immunoassay Using VHHs to Detect Toxin

Immunoassay are performed using VHH camelids to detect toxin in samples.Each of toxin-specific VHHs SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO:100,SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, and SEQ ID NO:162 are separatelyincubated in buffer in wells of a plastic microtiter plate. The VHHcamelids are incubated for sufficient time and under conditions suchthat the VHHs are adsorbed to the surface of the well. Control cells areincubated with buffer only.

A panel of diluated aliquots of a sample containing either a Shigatoxin, a B. anthracis toxin, a ricin A chain toxin, or a ricin B chaintoxin are incubated in duplicate in the VHH-coated wells and controlwells, such that the VHH in the VHH-coated wells specifically bind tothe toxin, thereby retaining the toxin in the well. Wells are washed toremove toxin that is not specifically bound to the VHH camelids.

A polyclonal antibody with enzymes or dye molecules attached to thepolyclonal antibody is contacted to the wells, thereby forming an toxinantigen ‘sandwich’ between the VHH camelids and the polyclonal antibody.The enzymes or dye molecules attached to the polyclonal antibodiesgenerate a color signal proportional to the amount of target toxinpresent in the sample added to the wells of the plate. It is observedthat the toxin-specific VHHs specifically bound the respective toxin,such that SEQ ID NO: 96 and SEQ ID NO: 98 specifically bind the Shigatoxin, and SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, and SEQ ID NO: 122specifically bind the anthrax toxin, and SEQ ID NO: 124, SEQ ID NO: 126,SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ IDNO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, and SEQ ID NO:144 specifically bind the ricin A chain toxin, and wherein SEQ ID NO:146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, and SEQ ID NO: 162specifically bind the ricin B chain toxin.

Example 34: Immunofluorescence Staining Using the Toxin-Specific VHHs

Subconfluent test cells on coverslips are treated with toxin (either aShiga toxin, a B. anthracis toxin, a ricin A chain toxin, or a ricin Bchain toxin) alone or toxin in the presence of the toxin-specific VHHs(specific VHHs SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:100, SEQID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110,SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ IDNO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138,SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ IDNO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQID NO:158, SEQ ID NO:160, and SEQ ID NO:162). The cells are fixed withparaformaldehyde, followed by permeabilization in a permeabilizingbuffer. For immunocomplex or toxin staining, cells are incubated withfluorochrome-conjugated anti-VHH, or polyclonal rabbit anti-toxin serum(prepared herein by methods known to one of skill in the art of antibodyproduction), followed by fluorochrome-conjugated anti-rabbit-IgG. Cellsare counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and imagedusing a confocal microscope. Surface binding of toxin-specific VHHs tocells is examined by flow cytometry.

Data from the immunofluorescence staining that the toxin-specific VHHsspecifically bind the respective toxin, and the toxin-specific VHHs areeffective for detecting the toxin, such that SEQ ID NO: 96 and SEQ IDNO: 98 specifically bind the Shiga toxin, and SEQ ID NO: 100, SEQ ID NO:102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:120, and SEQ ID NO: 122 specifically bind the anthrax toxin, and SEQ IDNO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132,SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ IDNO: 142, and SEQ ID NO: 144 specifically bind the ricin A chain toxin,and wherein SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, andSEQ ID NO: 162 specifically bind the ricin B chain toxin.

Test cells are incubated with toxin alone, toxin/toxin-specific VHH, ortoxin/non-specific VHH, followed by phycoerythrin-conjugated anti-VHHstaining. Cells are subsequently analyzed by cell sorting using a FACSCalibur flow cytometer. Data show that the VHHs effectively detect thelocation and relative abundance of specific Shiga toxin proteins,anthrax proteins, and ricin toxin proteins.

Example 35: Construction and Expression of a Heterotetramer of FourVHHs, Two Neutralizing VHHs that Target TcdA and Two VHHs that TargetTcdB

An embodiment of the invention herein provides small binding agents thatneutralize one of the C. difficile toxins have been identified. Thebinding agents are Vh domains prepared from heavy chain-only antibodiesof Camelid animals such as alpacas, also called VHHs. U.S. patentapplication Ser. No. 13/566,524 filed Aug. 3, 2012 and U.S. provisionpatent application No. 61/809,685 filed Apr. 8, 2013, which are herebyincorporated by reference in their entirety, describe proteins that havetwo linked pathogen neutralizing VHHs that recognize distinct,non-overlapping epitopes on the target, and those proteins have enhancedsubstantially enhance neutralization potency compared to each VHHseparately. Example 24 herein envisions linking neutralizing VHHs into aheteromultimer to target more than one pathogen, thereby permitting theneutralization of multiple pathogens by a single biomolecule. Thepresent example describes construction and expression of aheterotetramer of four VHHs, two neutralizing VHHs that target TcdA andtwo VHHs that target TcdB and was demonstrated to be active. Thismolecule, a VHH-based neutralizing agent (VNA) which targets both C.difficile Tcd toxins, is referred to as ‘VNA2-Tcd’.

The amino acid sequence of VNA2-Tcd protein is shown below (SEQ ID NO:170). The protein contains two copies of an epitopic tag, E-tag(GAPVPYPDPLEPR; SEQ ID NO: 15) that flanks the four linked recombinantVHH binding domain proteins and permits binding an anti-E-tag mAb to thetarget to promote antibody effector activities (see U.S. Pat. No.8,349,326 issued Jan. 8, 2013). The epitopic tag is optional and thissequence is merely exemplary and not further limiting. The sequencefurther includes an optional 13 amino acid albumin binding domain(DICLPRWGCLWED; SEQ ID NO: 168) at the carboxyl terminus to improveserum persistence of the VNA. See, Nguyen et al, 2006, ProteinEngineering, Design and Selection, 19:291. The VNA2-Tcd contains also anoptional amino terminal E. coli thioredoxin protein to improve proteinfolding and levels of soluble expression. The sequence was derived fromthe expression vector, pET32b. A thrombin and enterokinase cleavage sitewas introduced between the thioredoxin and the functional VNA to permitseparation of the two domains following expression of the product. Thevector was designed to separate the VHH proteins (underlined) by aflexible spacer to promote independent folding of each of the distinctVHH proteins.

Several amino acid modifications were made to the VHH protein sequencesas originally obtained and described in U.S. patent application Ser. No.13/566,524 filed Aug. 3, 2012 and U.S. provision patent application No.61/809,685 filed Apr. 8, 2013 to improve the framework region near theamino ends and improve protein folding and function (the amino terminalcoding region is typically modified during the VHH cloning process, andthe changes can be deleterious). Codons were optimized for improvedexpression in E. coli cells and regions of high DNA sequence homologywere modified to reduce the homology and thus reduce the likelihood ofDNA recombination. SEQ ID NO: 169 shows the coding DNA for the expressedVNA2-Tcd.

The VNA2-Tcd protein was expressed in E. coli and was purified usingNi-affinity chromatography followed by gel filtration chromatography(FIG. 44). Excellent purity and yields of soluble protein were observed.The purified VNA2-Tcd protein was demonstrated to be functional andbound to both C. difficile toxins, TcdA and TcdB, with high affinity inELISAs and displayed sub-nM IC₅₀ neutralization potencies specific forboth TcdA and TcdB in cell-based assays (FIG. 45). To test for in vivoefficacy, a C. difficile toxin systemic mouse challenge study wasperformed. Six week old female C57BL/6 mice were treated, viaintraperitoneal (IP) injection, with 50 ug/mouse of purified VNA2-Tcdone hour prior to IP challenge with 100 ng/mouse of C. difficile toxin A(TcdA) and 200 ng/mouse of C. difficile toxin B (TcdB). Control micechallenged with TcdA and TcdB all died or became moribund within 4 hourspost challenge. Untreated, VNA2-Tcd alone, TcdA+VNA2-Tcd, TcdB+VNA2-Tcd,and TcdA+TcdB+VNA2-Tcd treated animals showed no signs of systemiceffects and survived until study termination at 7 days post challenge(FIG. 46). These data demonstrate that VNA2-Tcd protein preventedlethality from intoxication by both TcdA and TcdB toxins. VNA2-Tcdprotein was demonstrated to be efficacious in a mouse model of C.difficile infection (CDI) (FIG. 47). Mice were treated with anantibiotic cocktail delivered in their drinking water for 3 days andthen treated with a single IP injection of clindamycin one day prior toinfection. Mice were infected with 10⁶ C. difficile UK6 spores alone orUK6 spores plus 3 doses of VNA2-Tcd protein (2.5 mg/kg at 4, 24 and 48hours after infection). Animals that received only the UK6 spores lostweight, had diarrhea, and 60% were moribund by Day 3. In contrast,animals that received treatment with VNA2-Tcd protein displayed minimalor no weight loss, did not have diarrhea after Day 1, and 100% of theanimals survived for the duration of the study. These data demonstratethe efficacy of VNA2-Tcd protein delivery as a therapy for CDI. Theefficacy of VNA2-Tcd was evaluated in a large animal model of CDI, thegnotobiotic pig model. For this study, gnotobiotic piglets were derivedvia Cesarean section and maintained in sterile isolators for theduration of the experiment. Cohorts of 6, 5 day old piglets were orallyinoculated with 10⁶ C. difficile UK6 spores only (control group), orwere administered VNA2-Tcd (1 mg/pig, IP) 4 hours prior to oralinoculation with spores (treated group). After the initial dose, thetreated group received 2 doses of VNA2-Tcd (1 mg/pig) each day for theduration of the experiment. Three out of six control pigs were moribundwith signs of weakness, lethargy, severe diarrhea and severe edematousrectal prolapse (Table 6). All pigs in the control and treated groupsdeveloped diarrhea within 48 hrs of inoculation with spores, howevernone of the VNA2-Tcd treated pigs became moribund, or developed rectalprolapse, and diarrhea was only mild to moderate in this group (Table6). All control piglets had signs of extra-intestinal lesions includingpleural effusion and ascities (Table 6). In contrast, no VNA2-Tcdpiglets showed any signs of ascities or pleural effusion (Table 6).

TABLE 6 Clinical outcome in VNA2-Tcd-treated and untreated C.difficile-infected piglets Treatment Clinical symptoms UK6 spores onlyVNA2-Tcd + UK6 spores Diarrhea^(a) 100 100 Mild diarrhea^(a) 0 16Mild/Moderate diarrhea^(a) 0 66 Moderate diarrhea^(a) 66 16 Severediarrhea^(a) 33 0 Rectal prolapse 83 0 Ascites 100 0 Pleural effusion 500 Edema 100 100 Systemic Disease^(b) 83 0 Fatal disease^(c) 50 0^(a)Severity of gastrointestinal disease was determined by clinicalsigns and histopathologic lesions, ranging from mild to severe^(b)Systemic disease indicates that piglets developed systemic signssuch as lethargy, weakness, anorexia, or dyspnea ^(c)Fatal diseaseindicates that piglets were euthanized due to the severity of thedisease

Additional gastrointestinal lesions in the severely affected pigletsinclude profound thickening of the wall of the colon and rectum, whichwas minimal or absent in treated piglets (FIG. 48). Histopathologicanalysis showed edema and lesions in both the control and treated groupswith primary differences seen in levels of mesocolonic edema andneutrophil infiltration in the distal colon (FIG. 49). Control pigs hadstatistically significantly more neutrophil foci in the lamina propriaof the distal colon compared to treated pigs. Control pigs had severemicrovilli degradation and neutrophil infiltration, while treated pigshad reduced microvilli degradation compared to controls and lessneutrophil infiltration (FIG. 49). These data demonstrate the efficacyof VNA2-Tcd protein for the treatment of CDI in a large animal model ofthe disease.

VNA2-Tcd coding DNA was inserted into a mammalian expression vector. Theprotein was observed secreted in the conditioned medium of the cells,and the product was observed to have retained full biologicalactivities.

The amino acid sequence of the full translation product expressed asVNA2-Tcd:

(SEQ ID NO: 170) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSGAPVPYPDPLEPRAAAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSAAAQLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSALVGGGGSGGGGSGGGGSLQAMAAAQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQAIAGGGGSGGGGSGGGGSLQGQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQGAPVPYPDPLEPRGGGSDICLPR WGCLWED

The coding sequence of VNA2-Tcd:

(SEQ ID NO: 169) ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGCCATGGCGATATCGGATCCGAATTCTGGCGCACCTGTCCCATACCCAGACCCTCTGGAACCACGAGCGGCCGCCCAAGTCCAACTGGTCGAAAGTGGTGGTGGTCTGGTCCAACCGGGTGGCTCTCTGCGTCTGTCCTGCGCTGCGAGTGGTTTTACCCTGGATTATAGCTCTATTGGTTGGTTCCGCCAGGCGCCGGGTAAAGAACGTGAAGGCGTGAGCTGCATCAGTTCCTCAGGTGATAGTACCAAATATGCGGACTCCGTCAAAGGCCGCTTTACCACGAGTCGTGATAACGCCAAAAATACGGTTTACCTGCAGATGAACTCCCTGAAACCGGATGACACCGCAGTGTATTACTGCGCGGCCTTTCGCGCTACGATGTGTGGTGTTTTCCCGCTGAGCCCGTATGGCAAAGATGACTGGGGTAAAGGCACCCTGGTGACGGTTTCGAGCGAACCGAAAACCCCGAAACCGCAGCCGACGTCTGCGATCGCCGGTGGTGGTGGTTCGGGTGGTGGTGGTAGCGGTGGTGGTGGTTCTGCAGCTGCGCAGCTGCAACTGGTGGAAAGCGGCGGTGGTCTGGTTCAACCGGGTGGTTCCCTGCGTCTGTCATGCGAAGCCTCGGGTTTTACCCTGGATTATTACGGTATTGGTTGGTTCCGTCAGCCGCCGGGCAAAGAACGTGAAGCAGTGAGCTATATTTCCGCATCAGCACGTACCATCCTGTACGCAGATTCAGTTAAAGGCCGCTTTACGATCTCGCGTGACAACGCGAAAAATGCCGTCTATCTGCAGATGAACAGTCTGAAACGTGAAGATACCGCAGTGTATTACTGTGCTCGTCGCCGTTTCTCCGCGTCTAGTGTCAATCGCTGGCTGGCCGATGACTACGATGTGTGGGGTCGTGGCACCCAAGTCGCCGTGTCCTCAGAGCCTAAAACGCCGAAACCGCAAACGTCTGCACTAGTTGGCGGTGGTGGCTCAGGTGGAGGCGGGTCAGGCGGTGGCGGTTCCCTGCAAGCAATGGCCGCAGCTCAGGTGCAACTGGTTGAATCCGGTGGTGGTCTGGTGCAGACCGGTGGTAGCCTGCGTCTGTCTTGCGCATCGAGCGGTAGCATTGCTGGCTTTGAAACCGTTACGTGGTCTCGTCAAGCGCCGGGTAAATCACTGCAGTGGGTCGCCTCGATGACCAAAACGAACAATGAAATCTATTCGGATAGCGTTAAAGGCCGCTTTATTATCTCACGCGATAACGCGAAAAATACCGTGTATCTGCAGATGAACTCGCTGAAACCGGAAGATACGGGTGTTTACTTCTGCAAAGGCCCGGAACTGCGCGGTCAAGGCATTCAGGTTACCGTCTCTAGTGAGCCTAAAACCCCGAAACCGCAAGCAATCGCAGGCGGCGGCGGCAGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCCCTGCAGGGTCAAGTCCAGCTGGTGGAATCTGGCGGTGGTCTGGTGCAACCGGGTGGTAGTCTGCGTCTGTCCTGTGCAGCCTCAGGCTTTACCTTCTCAGATTATGTTATGACGTGGGTCCGTCAGGCACCGGGTAAAGGTCCGGAATGGATTGCTACCATCAATACGGACGGTAGCACCATGCGCGATGACTCTACCAAAGGCCGCTTCACGATTAGCCGTGATAATGCCAAAAATACCCTGTACCTGCAGATGACGTCTCTGAAACCGGAAGACACCGCGCTGTATTACTGTGCCCGCGGTCGTGTTATTTCTGCAAGTGCTATCCGTGGCGCCGTCCGTGGTCCGGGCACCCAAGTCACCGTCTCCTCAGAACCGAAAACGCCGAAACCGCAACCGGCGCGCCAGGGTGCGCCGGTGCCGTATCCGGACCCGCTGGAACCGCGTTAA.

The amino acid sequence of the full translation product of mammaliancell secreted VNA2-Tcd:

(SEQ ID NO: 167) METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLSGAPVPYPDPLEPRAAAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSAAAQLQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSALVGGGGSGGGGSGGGGSLQAMAAAQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQAIAGGGGSGGGGSGGGGSLQGQVQLVESGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTIVIRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQGAPVPYPDPLEPRGGGSDICLPRWGCLWED.

Example 36: Dimer and Tetramer Constructs for C. difficile

In this example, the structure of the VNA multimers was modified toenhance resistance to GI proteases and improve therapeutic efficacy.

Potent, camelid C. difficile toxin binding single chain antibodies,VHHs, were developed to neutralize C. difficile toxins. To develop anorally-deliverable protein therapy for the treatment of C. difficileinfection (CDI), the C. difficile toxin-binding protein therapeuticshould persist within the GI tract for sufficient time to neutralize thetoxin. However, the GI track of mammals contains an abundance ofprotease enzymes used in food digestion that can degrade and inactivateorally delivered therapeutic proteins. The C. difficile toxin-bindingproteins (VHHs) display unexpected potent neutralizing activity whenjoined together in one molecule to form heterodimers andheterotetramers. Initial studies using GI tract extracts from pigsdemonstrated that the C. difficile toxin-binding proteins, whendelivered in heterodimeric and heterotetrameric forms, were rapidlydigested into monomers.

To determine the sites of cleavage in the heterodimeric andheterotetrameric C. difficile binding proteins, proteins were exposed toextracts of the porcine GI tract for different amounts of time, theproducts resolved by SDS-PAGE, purified, and subjected to amino-terminalsequence analyses. Sequencing revealed that the VHH functional domainswere resistant to cleavage, while the sites sensitive to cleavage werewithin the unstructured region(s) flanking the flexible spacer((GGGGS)₃) that connect the VHH domains together. This was an unexpectedfinding as the cleavage sites identified in this study do not representthose of known proteases, and were not predicted by proteomic analyses.Here, the identified sites contain one or more alanine residues,especially several adjacent alanines.

Without wishing to be bound by theory, to test the hypothesis thatmodification of the amino acid sequence flanking the flexible spacer((GGGGS)₃) would reduce proteolytic cleavage of the heteromeric proteinsin the GI tract, new proteins were designed and produced, VNA-TcdA(toxin A binding heterodimer, AH3+AA6; Seq ID NO: 171) and VNA-TcdB(toxin B-binding heterodimer, 5D+E3, Seq ID NO: 172), where alanineresidues were not included within the regions flanking the flexiblespacer ((GGGGS)₃) linking the VHH domains. The new dimer VNAs werecompared directly to the heterotetrameric VNA, VNA2-Tcd, that containsthe original, protease-sensitive regions (Seq ID NO: 167). VNA2-Tcdcontains the same four VHHs present in the new dimer VNAs, VNA-TcdA(AH3+AA6) and VNA-TcdB (5D+E3), joined into a heterotetramer(AH3+5D+E3+AA6). FIG. 50 shows that new dimers were highly resistant tocleavage by proteases present in the pig GI tract, while the tetramerwas rapidly cleaved to VHH monomers. These data demonstrate, amongothers, that the VNA multimers contained unexpected protease-sensitivesites and that modification of the sequence to remove these sitesresulted in resistance to digestion by GI tract proteases.

Example 37: Removal of Protease-Sensitive Sites Did not Affect C.difficile Toxin Binding Activity of Dimeric VNAs

The C. difficile toxin A-binding VHH dimer, VNA-TcdA, and toxinB-binding VHH dimer, VNA-TcdB, both containing the protease-resistantregion connecting the VHH monomers, were compared directly to thetetramer, VNA2-Tcd, that contains the protease-sensitive spacer regionsconnecting the VHH monomers, in an ELISA (FIG. 51). The tetramer anddimers showed similar affinities to the C. difficile toxins A (TcdA) ortoxin B (TcdB). Therefore, the sequence modification of the dimers hadno effect on toxin binding activities. Therefore, removal of theprotease-sensitive sites resulted in multimers with increased resistanceto GI proteases (FIG. 50) without loss of functional activity. Suchimproved protease-resistant multimers are anticipated to achieve greaterpersistence in the GI tract, improve the efficiency of C. difficiletoxin neutralization, and result in improved therapy for C. difficileinfection.

1.-18. (canceled)
 19. An isolated recombinant binding protein comprisingan amino acid sequence which is at least 95% identical to an amino acidsequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104,SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ IDNO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQ ID NO:122, which specifically binds to Anthrax protective antigen (PA) toxin.20. The isolated recombinant binding protein of claim 19, comprising anamino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ IDNO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112,SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQID NO: 122, which specifically binds to Anthrax protective antigen (PA)toxin.
 21. A composition comprising at least one recombinant bindingprotein that specifically binds to Anthrax protective antigen (PA)toxin, wherein the at least one binding protein comprises an amino acidsequence which is at least 95% identical to an amino acid sequenceselected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQ ID NO:
 122. 22. Thecomposition of claim 21, wherein the at least one binding proteincomprises an amino acid sequence selected from SEQ ID NO: 100, SEQ IDNO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ IDNO: 120, or SEQ ID NO:
 122. 23. A composition comprising at least onerecombinant binding protein that specifically binds to Anthraxprotective antigen (PA) toxin, wherein the at least one binding proteinis encoded by a polynucleotide sequence comprising a nucleotide sequenceselected from SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO:107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO:
 123. 24. Thecomposition of claim 21, further comprising a pharmaceuticallyacceptable carrier, excipient, or vehicle.
 25. The composition of claim22, further comprising a pharmaceutically acceptable carrier, excipient,or vehicle.
 26. A multimeric binding protein comprising at least tworecombinant camelid heavy-chain-only antibody monomers (VHHs) whichspecifically bind Anthrax protective antigen (PA) toxin and whichcomprise an amino acid sequence that is at least 95% identical to anamino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ IDNO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112,SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQID NO: 122; wherein the at least two VHHs are separated by spacers. 27.The multimeric binding protein of claim 26, wherein the VHHs comprise anamino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ IDNO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112,SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQID NO:
 122. 28. The multimeric binding protein of claim 26, comprisingtwo VHHs which bind Anthrax PA toxin.
 29. The multimeric binding proteinof claim 26, further comprising one or more epitope tags to which ananti-epitope tag antibody specifically binds.
 30. The multimeric bindingprotein of claim 26, wherein the spacer comprises a peptide having anamino acid sequence selected from GGGGS (SEQ ID NO: 54), GGGGSGGGGSGGGGS(SEQ ID NO: 55), a combination thereof, or a nucleotide sequenceencoding said amino acid sequence.
 31. The multimeric binding protein ofclaim 29, wherein the one or more epitope tags comprises the amino acidsequence Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg of SEQ IDNO: 15, or a nucleic acid encoding said amino acid sequence.
 32. Apharmaceutical composition comprising the multimeric binding protein ofclaim 26 and a pharmaceutically acceptable carrier, excipient, orvehicle.
 33. A pharmaceutical composition comprising the multimericbinding protein of claim 27 and a pharmaceutically acceptable carrier,excipient, or vehicle.
 34. A method of neutralizing Anthrax protectiveantigen (PA) toxin, said method comprising contacting said PA toxin withthe composition of claim 24 in an amount effective to neutralize theAnthrax PA toxin.
 35. The method according to claim 34, wherein theAnthrax PA toxin is neutralized in vitro or in vivo.
 36. A method ofneutralizing Anthrax protective antigen (PA) toxin, said methodcomprising contacting said PA toxin with the composition of claim 25 inan amount effective to neutralize Anthrax PA toxin.
 37. The methodaccording to claim 36, wherein the Anthrax PA toxin is neutralized invitro or in vivo.
 38. A method of neutralizing Anthrax protectiveantigen (PA) toxin, said method comprising contacting said PA toxin withthe composition of claim 32 in an amount effective to neutralize AnthraxPA toxin.
 39. The method according to claim 38, wherein the Anthrax PAtoxin is neutralized in vitro or in vivo.
 40. A method of neutralizingAnthrax protective antigen (PA) toxin, said method comprising contactingsaid PA toxin with the composition of claim 33 in an amount effective toneutralize Anthrax PA toxin.
 41. The method according to claim 40,wherein the Anthrax PA toxin is neutralized in vitro or in vivo.
 42. Amethod of treating or preventing intoxication of a subject by Anthraxprotective antigen (PA) toxin, said method comprising: administering toa subject in need thereof the composition of claim 24 in an amounteffective for the binding protein to bind to and neutralize Anthrax PAtoxin in the subject after intoxication or prior to the subject's havingsymptoms of intoxication.
 43. A method of treating or preventingintoxication of a subject by Anthrax protective antigen (PA) toxin, saidmethod comprising: administering to a subject in need thereof thebinding protein of claim 25, or a pharmaceutical composition thereof, inan amount effective for the binding protein to bind to and neutralizeAnthrax PA toxin in the subject after intoxication or prior to thesubject's having symptoms of intoxication.
 44. A method of treating orpreventing intoxication of a subject by Anthrax protective antigen (PA)toxin, said method comprising: administering to a subject in needthereof the composition of claim 32 in an amount effective for themultimeric binding protein to bind to and neutralize Anthrax PA toxin inthe subject after intoxication or prior to the subject's having symptomsof intoxication.
 45. A method of treating or preventing intoxication ofa subject by Anthrax protective antigen (PA) toxin, said methodcomprising: administering to a subject in need thereof the compositionof claim 33 in an amount effective for the multimeric binding protein tobind to and neutralize Anthrax PA toxin in the subject afterintoxication or prior to the subject's having symptoms of intoxication.